Translating Pre-Clinical Research to Clinical Patient Care™
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Plenary Presentations
Podium Presentations
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Immuno-MALDI for Accurate and Precise Clinical Protein Quantitation
Immuno-MALDI (iMALDI) technology combines the sensitivity of immunoaffinity capture with the specificity of mass spectrometry detection. We have now taken a multifaceted approach for translating iMALDI technology into clinical laboratories for routine protein quantification. First, we have automated the sample preparation using the Agilent Bravo liquid handling robot for improved sample throughput. Secondly, we have optimized iMALDI assays for the Bruker Microflex MALDI-TOF, a bench-top instrument that is widely used in regulated healthcare environments. We demonstrate that iMALDI technology is suitable for clinical use through the precise and accurate measurement of the plasma renin activity in >200 clinical samples.
Method Development with Easy to Automate Absorptive Chemistry Extraction for LC-MS
When using the innovative AC Extraction Plate(TM) for the extraction of small molecules, the method development process can be simplified due to the plate’s unique ‘pipette and shake’ workflow. Initial optimization simultaneously deals with three steps (extraction, wash and elution) in a matrix approach, the Direct Extraction-Elution Method (DEEM). This process evaluates the optimum conditions for the analyte(s) interaction with the absorptive chemistry (AC) of the extraction plate. Further optimization of conditions for the extraction step tackle the interaction of analyte with the sample matrix (e.g., protein binding).
For analytes such as steroids (e.g., Testosterone, Androstendione, Estradiol) the extraction conditions must be modified and adapted to overcome protein binding. Experience with the optimization of this critical step will be outlined and interesting examples discussed.
Rapid Bedside Diagnosis Tools by Coupling of Bio-compatible Solid Phase Microextraction (SPME) Devices to Mass Spectrometry
SPME is a green sample preparation technique that combines extraction and pre-concentration of analytes in one step thus simplifying the analytical process. Succinctly, SPME does not require any sample collection because extraction takes place in situ by inserting a biocompatible microfiber directly into tissue, blood or other biological matrix for a short period of time. Alternatively, the same device can be used for ex vivo analysis using a small amount of the studied sample. This work presents multiple strategies recently developed for the direct coupling of SPME to MS. In order to have a broader range of applications, different SPME geometries such coated fibers and meshes, as well as ionization approaches such DART and ESI, were studied.
Metabolic Profiling as a Tool for Investigating Diseases of Pregnancy
Metabolic profiling can offer insights into disease diagnosis, progression, and responses to therapeutic intervention. Ultra-performance liquid chromatography – mass spectrometry (UPLC-MS) is a powerful tool for metabolic profiling, with excellent separation and detection capabilities. We developed a robust, reproducible UPLC-MS method for placental extract profiling and applied this to a cohort of women with intrahepatic cholestasis of pregnancy (ICP). We demonstrated clear metabolic differences between treated and untreated ICP patients, which may be reflected in plasma/serum, offering the potential for a minimally invasive diagnostic tool. This approach could be extended to study other pregnancy complications, such as pre-eclampsia and pre-term delivery.
Quantitative Multiplex Assays for Inborn Metabolic Disorders in Dried Blood Spots
In recent years, more efficient therapies are becoming available for inherited lysosomal storage disorders (LSDs) and timely initiation of treatment often leads to a better outcome. Consequently, tandem mass spectrometry based screening assays in newborns are being increasingly implemented in clinical laboratories. We have been developing a fully quantitative assay of nine lysosomal enzymes in dried blood spots (DBS), which screens for Niemann-Pick-A/B, Pompe, Fabry, Gaucher, Krabbe and mocopolysacharidoses I; II; IVA and VI diseases. We will present our latest progress and future directions of LSDs assay development. Further, we will demonstrate the clinical value of data from LSDs pilot studies conducted by our collaborators and worldwide.
Study of Pregnancy Outcomes: Quantification of Selected Metabolites by High-throughput Nano-electrospray HRMS-TOF Method
The precise, accurate and rapid validated high-throughput direct injection nano-ESI-HRMS was applied for quantification of several selected metabolites in the urine samples of the female patients with different pregnancy outcomes (Pregnant, Non-Pregnant and Early Pregnancy Loss (EPL)). The absolute quantification of eight selected metabolites was achieved by standard addition using stable isotope labelled internal standards, using a composite urine sample to account for any matrix effect. Some other metabolites were quantified relative to these internal standards. The concentration of some metabolites showed differences for different pregnancy outcomes. The full-scan data was used for untargeted fingerprinting.
A New Method to Assess Sequencing & Annotation Quality in Databases Used for Clinical Proteomics and Metaproteomics
Advances in next-generation genome sequencing have made proteomic experiments more successful than ever. However, genome sequences are contaminated more frequently than is admitted, with large impact on most proteomics fields. Here, we propose a new concept to highlight abnormal organism sequencing data and quickly identify the source of contamination. A specific software program was developed for a quick spotting of cross-contamination of organisms, using specific experimental MS/MS data. We highlighted two likely contaminated WGS data detected in the NCBInr database and confirmed this discovery by large scale blast analysis. Other examples related to pathogens, and associated problems will be commented. Such new concept should rapidly improve the quality of sequence databases of upmost importance for clinical proteomics analysis.
Quality Assurance in Clinical Mass Spectrometry
Among the analytical techniques used in clinical pathology today, mass spectrometry based methods potentially enable analyses on a unique level or reliability: signal generation is based on molecular weight an disintegration patterns of analytes, and application of stable isotope dilution internal standardisation suggests complete compensation of individual matrix effects. However, high complexity of the technology, the dynamic nature of analyte ionisation, highly variable instrument configurations, and incomplete solutions for automation challenge the quality of mass spectrometric methods. In this presentation specific requirements for quality assurance of mass spectrometry methods applied in clinical diagnostics regarding patients’ safety are discussed.
Influence of Glycosylation for Providing Relevant “clinical” Protein Calibrants
Sound medical decisions rely on accurate clinical measurements. To be trustable and comparable, these measurement results have to be metrologically traceable which can be established by using protein-based certified reference materials (CRMs). Many clinical diagnostic methods for proteins are based on immunoassays. Since proteins undergo post-translational modifications such as glycosylation, possible changes in glycosylation might affect protein recognition and its quantitation by immunoassays. However, this effect has hardly been studied in available CRMs. This work aims to evaluate the impact of glycosylation on the commutability and value assignment of several protein-calibrants by liquid chromatography-mass spectrometry, capillary electrophoresis and circular dichroism.
TDM in Psychopharmacology Using LC-MS/MS – from the Complex Method to the Interpreted Result
Therapeutic Drug Monitoring (TDM) is an important tool for individualized psychopharmacotherapy, allowing to get a kind of ‘pharmacologic phenotyping’ of the patient.
From the methodological aspect, TDM in psychopharmacotherapy is facing one major problem: Polypharmacy, leading to the necessity to use a highly specific and at the same time robust and sensitive method. LC-MS/MS for TDM of psychotropic drugs is therefore more and more becoming the gold standard.
This presentation will address the major analytical, pharmacokinetic, pharmacogenetic and practical aspects of TDM in psychiatry to demonstrate how TDM can be used for individualized risk reduction.
CYP1A2 Phenotyping by Measuring Paraxanthine/caffeine Concentration Ratios in Hair and Comparison with the Plasma-based Phenotype
Measuring metabolite-to-parent drug concentration ratios in hair may provide a convenient tool to study drug metabolism in a non-invasive way. We evaluated whether paraxanthine/caffeine ratios measured in hair samples reflect the plasma-based CYP1A2 phenotype. Using a validated LC-MS/MS method, caffeine and paraxanthine concentrations were measured in proximal 3 cm segments of hair samples from 60 healthy volunteers and resulting paraxanthine/caffeine ratios were correlated with CYP1A2 phenotyping indices measured in plasma. Although paraxanthine/caffeine concentration ratios in hair and plasma showed overall a statistically significant correlation, large deviations between hair and plasma ratios in individual cases impede interpretation.
Ultra-low Level Clinical Analysis Using LC-MS/MS Technologies
Following the explosion of steroid hormone assays in the mid 2000's, advancements in techniques and analytical capabilities has enabled LC-MS/MS tools to supersede the performance characteristics of ELISA and RIA assays. We have leveraged these advancements to enable ultra-low level clinical biomarker analysis (<1pg/mL). This presentation will highlight the analytical challenges and solutions to realize these criterion. Specific assays for determination of estradiol/estrone (200fg/mL), free (equilibrium dialysis) and salivary Testosterone (500fg/mL), Thyroglobulin (1pg/mL peptide level) and free T3/T4 (1pg/mL)will be described. Further, multiplexing of these assays to <1500 samples/system/day will be shown.
MSIA Workflow for Comprehensive Identification and Analysis of Isobaric Insulins: An Approach to Targeted Insulin Quantitation Using HR/AM-MS and Multiplexed LC
There is a trend towards the analysis of insulins using LC-MS. We report a mass spectrometric immunoassay (MSIA) method, with high-resolution, accurate mass detection (HR/AM), and chromatographic resolution of a known isobaric insulin analogue, insulin lispro. Total analysis time was 15 minutes (without multiplexing). Analytes were detected using a Q Exactive MS (Thermo Scientific). Insulin, insulin lispro, and the internal reference standard are enriched simultaneously from samples using the insulin MSIA method. Combined with HR/AM, it is possible to acquire full-scan and MS2 data for both analytes independently, allowing (i) robust peak identification, and (ii) quantitative analysis of both compounds.
High Selectivity and Sensitivity in LC-MS Clinical Assays
New hybrid mass spectrometers with high resolution and accurate mass capabilities have opened new avenues in quantitative proteomics. Targeted clinical analyses, routinely performed on triple quadrupole instruments, were replicated on a high-resolution quadrupole-orbitrap instrument operated in parallel reaction monitoring (PRM) mode. The trapping capability was used to analyze peptides in tiny amounts, thus increasing the dynamic range while providing selective measurements. The PRM technique was applied to analyze makers in lung cancer markers. The gain in selectivity and an increase in the confidence of measurements in a clear discrimination of the disease stages and subtypes.
Identification of Novel Biomarkers of Brain Injury by Integrating Bioinformatics and Mass Spectrometry-based Proteomics
Hemorrhagic stroke (HS) is a significant cause of mortality worldwide. A blood-based diagnostic test to identify this condition would be useful. The aim was to develop selected reaction monitoring (SRM) assays to quantify “brain specific” proteins in CSF from patients with HS, ischemic stroke and controls. SRM assays for 68 proteins were developed. Six peptides from proteins GFAP, MBP, NFM, NSE, α-Inx and β-Syn were significantly elevated in the HS group. NFM was further evaluated using an ELISA. Serum NFM concentration in controls (n=46) was from 0.26 to 8.57 ng/mL, while in 78 serial samples from 7 patients with HS was from 0.97 to 42.4 ng/mL.
MALDI Molecular Imaging of Proteins, Metabolites and Drugs for Preclinical and Clinical Research
This presentation will give an update on the application of MALDI imaging in preclinical and clinical research. We discuss the use of MALDI imaging in clinical proteomics and put it in context with classical proteomics techniques for tissue analysis. In the research area of gastrointestinal disorders MALDI imaging has already been used to address several questions of upper- and lower gastrointestinal diseases, which will be briefly presented. We also highlight a number of upcoming challenges for personalized medicine, development of targeted therapies and diagnostic molecular pathology where MALDI imaging could help.
Spatial Metabolomics: Database-Driven Metabolic Annotation for High-resolution Imaging Mass Spectrometry
High-resolution Imaging Mass Spectrometry (imaging MS) is a promising technique for untargeted spatial metabolomics. We present a novel database-driven and high-throughput approach to generate hypotheses on metabolites represented in high-resolution imaging MS data. Rather than identifying molecular species for millions of individual peaks, we restricted each imaging dataset to signals potentially corresponding to molecules from metabolic databases. Sum formulas of all metabolites were considered and corresponding ion images were generated. For each sum formula, an annotation score was calculated which integrates various spatial and spectral characteristics. Hundreds of sum formulas were detected as corresponding to metabolites present in the tissue sections. The evaluation confirmed the potential of our approach to provide relevant hypotheses on metabolites present in a tissue section.
A New Approach to Biomarker Discovery in Clinical Mass Spectrometry Through Statistical Modelling of the Raw Data
We present a new type of workflow for differential analysis of LC-MS data in clinical discovery. The fundamental principle is to retain and model the raw data from start to finish, thus enabling detection below the limit of current software tools, and the assessment of differential expression in overlapping peptide signals. The data is analysed entirely in raw form, so that the full profile-mode MS1 signal is retained and utilised for differential quantification. Each LC-MS dataset is denoised and converted to an image, warped by a novel LC alignment stage and then statistical analysis performed directly on the set of aligned images through a Bayesian mixed-effects model. No error-prone peak detection or deconvolution is necessary. This enables our workflow to handle complex experimental designs with multiple experimental conditions, sources of variation and batch effects.
Immunoaffinity Extraction Coupled Online with LC-MS/MS for the Quantification of Total Plasma Testosterone: A Feasibility Study
Immunoaffinity extraction was coupled online with LC-MS/MS to explore the possibility of quantifying total plasma testosterone. 25 µL plasma was analyzed using immunoaffinity- or C8-sorbents. Extraction and elution parameters were optimized. Ion suppression experiment results were comparable between immunoaffinity extraction and C8-extraction. We showed that it is feasible to combine online immunoaffinity extraction with LC-MS/MS for the determination of total plasma testosterone. Currently we are combining antibodies that capture different compounds to enable the multiplex analysis of disease specific biomarker panels.
Towards a LC-MS/MS Based Clinical-chemical Analyzer
for Small Molecules in Body Fluids
For a broad implementation and routine application of LC-MS/MS in clinical laboratories, sample pretreatment has to be integrated and fully automated. Towards this, we developed a novel instrumental platform which – for the first time – enables a fully automated in-line processing not only of native blood plasma, blood serum, cerebrospinal fluid, saliva and urine but also of anticoagulated whole blood samples prior to on-line SPE-LC-MS/MS analysis. Perfusion through a heated stainless-steel capillary converts whole blood into so-called cell-disintegrated blood (CDB). CDB represents a homogenous liquid composed of subcellular particles which do not sediment on standing and do not clog LC-capillaries, sieves or column packings. Target analytes present in CDB and other biofluids are extracted by on-line SPE under high flow velocity conditions.
Potassium-based Algorithm Allows Correction for the Hematocrit Bias in Quantitative Analysis of Caffeine and Its Major Metabolite in Dried Blood Spots
Deviating Hct values may cause significant bias in quantitative dried blood spot (DBS) analysis. We evaluated whether a potassium-based algorithm allowed correction of the Hct bias, using caffeine as a model compound. An algorithm was constructed using data from a reference set of DBS and whole blood samples and applied to a separate test set. While at Hct levels below 0.36, caffeine concentrations in DBS were significantly underestimated compared with blood, this was no longer the case after application of the algorithm. The usefulness of this approach was further demonstrated by applying the same algorithm to paraxanthine, yielding similar results.
Investigating Beneficial Changes in Human Metabolism and Immunological and Inflammatory Markers Following High Intensity Interval Or Regular Endurance Training
Age-related declining health has a significant impact upon quality of life and healthcare costs. Exercise is a non-invasive intervention to maintain healthy status by reducing deleterious changes in immune and inflammatory status. Understanding the interactions between exercise, age and metabolism will provide insights in to how exercise interventions can maximise their effect in maintaining health as we age. The presentation will discuss a mass spectrometry metabolomics study of changes in the plasma metabolome related to two different exercise regimes, age and inflammatory/immunological status. The presentation will also discuss the importance of quality assurance and metabolite annotation in these discovery studies.
Spatial Metabolomics of Three-dimensional Cell Culture Systems
3D cell cultures of colon adenocarcinoma provide an advanced in vitro model for studying metabolic processes within tumours, and their response to drug treatment. However, system-wide analysis requires untargeted detection of metabolites inside these spheroid cultures and localization to a particular spheroid layer. We addressed this analytical challenge using our newly developed spatial metabolomics approach, which combines high mass-resolution imaging mass spectrometry with high-throughput database-driven molecular annotation of imaging mass spectrometry data. Our approach allowed us to detect hundreds of signals and transform the large high mass resolution data into an easily interpretable form for experts in cancer metabolomics.
Application of Oxylipin Profiling to a Sulindac Intervention of Pain.
Despite successful use of selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs) for the treatment of estrogen receptor positive (ER+) breast tumors, 25-30% of ER+ patients still die from their disease. High rates of early discontinuation of AIs (estimated at 30% by year 3) due to intolerance to side effects, notably musculoskeletal pain or AI-induced musculoskeletal syndrome, are now linked to reduced benefit. Here, within the context of an ongoing clinical trial using UPLC-QTRAP, we have profiled the biologically active oxylipin metabolites of Ω-6 and Ω-3 fatty acids in plasma and urine in order to understand their relationship to AI-induced pain as well as response to the pain-reducing drug, sulindac. Profiles were highly interconnected with ~1/3rd of the first set of 10 patients harboring a theoretical systemic pro-inflammatory metabolome.
Quantitation of Soluble Transferrin Receptor (sTfR) in Human Serum Using SISCAPA Immunocapture Enrichment and MALDI-TOF Mass Spectrometry
A sensitive, accurate, high-throughput and automated assay was developed for LC-free SISCAPA-MALDI quantification of sTfR. The traditional ELISA assay for quantitation of sTfR in human serum is suspected to be subject to protein interferences that can lead to inaccurate results. Here we present an alternative approach to the ELISA assay. This approach consists of automated digestion of the sample, including any present protein interferences, followed by automated, parallel analyte enrichment in 96-well format and quantitation using MALDI-TOF mass spectrometry. The approach was assessed for sensitivity and precision and was determined to be suitable for clinical analysis of patient samples.
Real-time Identification of Gastrointestinal Polyps and Other Alterations During Endoscopic Procedures: The iEndoscope
Endoscopic screening is routinely used for the early stage detection of gastrointestinal tumours. Our aim is to create a fast, in-situ endoscopic tissue identification tool based on rapid evaporative ionization mass spectrometry (REIMS). The iEndoscope method has been shown to be capable of differentiating between healthy mucosa, cancer, adenomatous polyps and other tissue degenerations based on the REIMS fingerprint of each tissue type. The novel iEndoscope is a feasible technique for rapid identification of human tissue in-vivo during endoscopic interventions, and it can also be used as a safety tool giving a warning signal if the submucosal region becomes damaged.
Rapid Characterization and Identification of Clinically Relevant Microorganisms Using Rapid Evaporative Ionization Mass Spectrometry
The capabilities of rapid evaporative ionization mass spectrometry (REIMS) as a general identification system for microorganisms are presented. Strains of 28 clinically relevant bacterial species were analyzed in negative ion mode and corresponding data subjected to unsupervised and supervised multivariate statistical analyses. The created supervised model yielded correct cross-validation results of 95.9%, 97.8% and 100% on species-, genus- and Gram-stain-level, respectively. Additionally, the technique proved suitable to distinguish five pathogenic Candida species with 98.8% accuracy without any further modification to the experimental workflow. Subspecies specificity is shown in case of seven Escherichia coli strains and three different Clostridium difficile ribotypes.
A Proteogenomic Strategy for Defining Biomarkers for Quick Identification of Francisella Subspecies by MALDI-TOF MS
The Francisella tularensis pathogen is the causative agent of tularemia and a potential bioweapon of category A due to its high virulence. By means of an original proteogenomics strategy relying on a large panel of genomic data on Francisella bacteria and the combination of shotgun proteomics and whole-cell MALDI-TOF mass spectrometry, we established that a unique set of three protein biomarkers could enable the identification of Francisella species and subspecies, thus predicting its virulence level. Their detection as intense peaks in the most virulent subspecies of Francisella confirmed the validity of this approach that could be extended to any pathogens.
Identification of Noninvasive Biomarkers of Coordinate Metabolic Reprogramming Associated with Colorectal Cancer
Tandem tissue and urinary metabolomic profiling revealed early noninvasive biomarkers of colorectal tumorigensis in ApcMin/+ mice. Transcriptomic analysis in ApcMin/+ and mice with colon-specific disruption of Apc gene showed that these biomarkers were reflection of coordinate reprogramming metabolic pathways including methylation during colorectal carcinogenesis. AOM-induced colorectal carcinogenesis mouse model showed that such metabolic reprogramming-associated biomarkers were a conserved feature of colorectal carcinogenesis in mice irrespective of etiology and genetic background. Analysis of paired non-tumor and tumor tissues from colorectal cancer patients showed that such metabolic derangements are also associated with human colorectal carcinogenesis in stage-dependent manner.
Touch Spray Mass Spectrometry (TS-MS) Used for Rapid Diagnosis of Kidney and Prostate Cancer Using Tissue Specimen Obtained from Surgery
Touch spray uses a small probe to pick up sample and an application of voltage and solvent to cause field-induced droplet emission for MS analysis. TS was used in a study of 18 prostatectomy cases to differentiate prostate cancer and healthy tissue with 95% accuracy based on initial cross validation results of the first 12. A blind validation was performed on the latter six patients to confirm TS as a possible in vivo surgical tool. For a professional assessment of TS-MS, Dr. Timothy Masterson performed the technique in vitro on a radical nephrectomy specimen targeting diseased and normal tissue.
Rapid Quantification of Cortisol, Cortisone, Dexamethasone and Prednisolone in Human Saliva and Hair by Liquid Chromatography – Tandem Mass Spectrometry
Reliable determination of cortisol and cortisone concentration in saliva and hair is of interest in the assessment of stress-associated adrenocortical function. To this purpose we developed a robust method for the reliable, simultaneous quantification of glucocorticoids via LC-MS/MS, overbearing the shortcomings of immunological analysis. With excellent precision (2-7%), very good recovery rates (95-115%) and a total runtime of 4.5 min we proved the benefits of LC-MS/MS over immunoassays. Moreover, we analysed 2500 samples of the LIFE Child Depression study and demonstrated the massive impact of preanalytical factors on hair analysis, enabling proper evaluation in clinical routine diagnostics and epidemiological studies.
Profiling Thiol Metabolites and Quantification of Cellular Glutathione Using FT-ICR-MS
We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue *QDE as reagents for chemoselective derivatization and analysis of cellular thiols using FT-ICR-MS. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method was readily applied to quantify the thiol metabolites glutathione and glutathione disulfide in A549 cells and the concentrations were found to be 34.4 ± 11.5 nmol/mg protein and 10.1 ± 4.0 nmol/mg protein, respectively.
A Protective Lipidomic Biosignature Associated with a Balanced Omega-6/omega-3 Ratio in Fat-1 Transgenic Mice
A balanced omega-6/omega-3 polyunsaturated fatty acid (PUFA) ratio has been linked to health benefits and the prevention of many chronic diseases. Current dietary intervention studies with different sources of omega-3 fatty acids (omega-3) lack appropriate control diets and carry many other confounding factors derived from genetic and environmental variability. In this study, we used a multi-platform lipidomic approach to phenotype the molecular phenotype of plasma samples from an animal model of long term omega-3 supplementation. Integration of the results of untargeted and targeted analyses has identified a lipidomic biosignature that may underlie the healthful phenotype associated with a balanced omega-6/omega-3 ratio, and can potentially be used as a circulating biomarker for monitoring the health status and the efficacy of omega-3 intervention in humans.
Five Years of Urinary Screening for Drugs of Abuse by Tandem Mass Spectrometry and the Evolution to Pain Management Testing for Compliance and Diversion
We have been providing high volume drugs of abuse screening by tandem mass spectrometry for over 40 different drugs/metabolites to clinicians for more than five years. For the last few years, physicians have been asking for changes to our program, which would provide them improved information about low dose opioid/synthetic pain medication usage, compliance and possible diversion detection. This is logical given that the greatest increase in drug misuse is of prescription drugs; specifically pain medications. The major obstacles to this request were establishing lower, yet reliable cut-off values and providing quantitative data over a very broad dynamic range.
Detection of Allenic Norleucine, a Nephrotoxic Amino Acid from Amanita Smithiana and Related Mushrooms
Amanita smithiana is a poisonous mushroom, causing acute renal failure, that grows along the Pacific coast of North America. Several European Amanita species are thought to share this same toxin, a non-protein amino acid called allenic norleucine. Previously, thin layer chromatography was the method used to confirm presence of the toxin, however, this was labour-intensive and subjective in its interpretation. We have now developed an LC-MS/MS method for allenic norleucine. Apart from its potential clinical utility in poisoning cases, we have used the assay to screen a large number of mushroom species for the toxin.
On the Possibility of Using Exhaled Breath for Toxicological Investigations
Exhaled breath has recently been proposed as a matrix for drug testing. A serie of investigations have demonstrated that most common drugs of abuse are detectable in breath following intake. The normal breathing process creates aerosol micro-particles that are exhaled in breath. These patrticles are formed from the airway lining fluid during the normal breathing process. The airway lining fluid can become contaminated with drugs present in the body. These aerosol particles constitutes a way for non-volatile compounds to exit lung.
This presentation will give an update on the present status in the field of drug testing in exhaled breath.
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Poster Presentations
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Analysis of Amphetamines in Urine Sample Using the Atmospheric-pressure Solids Analysis Probe for Ionization
Analysis of amphetamines in urine sample using the atmospheric-pressure solids analysis probe for ionization
Fernanda Helena Salami, Eduardo José Crevelin and Luiz Alberto Beraldo de Moraes
Stimulants are substances capable of reducing tiredness and increasing alertness, competitiveness and aggression because of these effects. They are used by drivers and also as appetite suppressant for weight reduction or in sports competition and training to increase athlete performance. Here, atmospheric-pressure solids analysis probe for ionization coupled to MS/MS (ASAP-MS/MS) was utilized in the analysis of amphetamines and cocaine in urine samples. Limits of detection for stimulants were determined 1ng mL-1. ASAP-MS/MS was rapid and sensitive technique with potential for determination of amphetamines and cocaine in urine simple.
Quantitative Target Metabolomics Using LCMS/MS Improves the Diagnosis of Vitamin B12 Deficiency and Saves Huge Cost and Time
Early diagnosis of vitamin B12 deficiency can prevent irreversible complications. Plasma metabolite, methylmalonic acid (MMA) and active-B12 have made their way into modern routine laboratories to replace the traditional assay of total B12 determination. MMA was measured in a 2-step-algorithm after total or active-B12 in 1034 samples. MMA analysis reduced false negative results that were produced using total or active-B12 alone. Plasma MMA levels were explained by diet, renal function and supplementation. A new LC-MS/MS assay for MMA is comparable to the reference GC-MS assay. It is considerable cheaper and takes up less time from sample preparation to reporting results.
Proteomics and Biomarkers in Osteoarthritis
The application of proteomic techniques to disease states affords the opportunity to identify deregulated pathways that contribute to pathogenesis. These discoveries in turn may lead to new therapeutic targets, as well as biomarkers that aid in diagnosis, prognosis and the prediction of treatment responses. Osteoarthritis (OA) is a disease of all joint structures and synovial fluid (SF) may represent of synthesis of inputs from these structures. We compared the proteomic profile of knee joint SF from patients with early and late stage OA to unaffected controls by mass spectrometry. In our recent publication, 66 proteins were reported as differentially represented in healthy vs. OA. Pathway analysis identified three biologic processes among these proteins: the complement and coagulation systems and the acute phase response. Early and late OA manifested a very similar proteomic profile.
Performance of Symmetric Dimethylarginine Against Other Markers of Kidney Function
Glomerular filtration rate (GFR) is the best overall index of kidney function. Knowledge of GFR is essential to the diagnosis, classification, and management of kidney disease. Serum measurements of cystatin C (cysC), creatinine and their equations are the most widely used indirect markers, while symmetric dimethylarginine (SDMA) is an emerging marker. The objective of this study was to compare the performance of SDMA with cysC, creatinine and their equations to direct GFR measurement by radioactive iothalamate. A total of 40 subjects were included in this study. A published LC-MS/MS method was used for measuring SDMA. CysC and creatinine were measured by Roche Cobas 8000 (Indianapolis, IN). CysC showed the best overall correlation with GFR (r=0.92) and highest AUC (0.923) for kidney donor eligibility, followed by SDMA (r=0.87, AUC=0.882) then creatinine (r=0.76, AUC=0.767).
Sensitive 2D-UHPLC-MS/MS-Method for Simultaneous Quantification of Seven Corticosteroids to Investigate Adreno-cortical Dysfunction in Critically Ill Patients
The role of adreno-cortical dysfunction in septicaemia is a widely discussed topic in clinical research worldwide. In order to improve the biochemical description of this condition a highly sensitive and very selective 2D-UHPLC-MS/MS-method was developed addressing cortisol, cortison and aldosterone as well as their four precursors (corticosterone, 11-desoxycortisol, 17-hydroxyprogesterone, 11-desoxycorticosterone). Since large clinical studies require a high-throughput method we decided to apply two-dimensional chromatography with protein precipitation as the only manual step of sample preparation.
Determination of Insulin-Like Growth Factor-1 in Plasma by HRAM LC/MS for Clinical Research
High Resolution Accurate Mass (HRAM) Liquid Chromatography Mass Spectrometry (LC/MS) is ideally suited for the rapid analysis of biomolecules. A highly sensitive and specific HRAM LC/MS method has been developed for the quantitation of Insulin-Like Growth Factor-1 (IGF-1). This method uses a simple sample preparation combined with an online sample cleanup procedure coupled to a high resolution accurate mass quadrupole time-of-flight mass spectrometer. The described method achieves the required functional sensitivity and is capable of quantitating IGF-1 over a sufficiently wide dynamic range at R2 > 0.999. Intra- and inter- day CVs are below 5 and 10% respectively.
New LC-MS/MS Method for the Determination of Total Homocysteine in Whole Blood (Dried Blood Spots) and Serum/Plasma
A LC-MS/MS method was developed allowing now the determination of total homocysteine (tHcy) in Dried Blood Spots (DBS) and in plasma/serum as well.
While the LC-MS/MS method is the same, there are just slight changes in sample preparation for both matrices. It includes reduction of protein-bound or dimerized homocysteine, followed by extraction for DBS samples and protein precipitation for plasma/serum samples, respectively.
With an injection interval of only 1 minute, the fast, easy and reliable determination of tHcy in DBS and Plasma/Serum is now possible.
A Rapid Clinical Research Method for the Simultaneous Analysis of Plasma Catecholamines and Metanephrines by Mixed-Mode SPE and HILIC LC/MS/MS
An analytical method was developed for plasma catecholamines and metanephrines for clinical research purposes. Analytes extracted using weak cation exchange SPE were analyzed using HILIC LC/MS/MS. Extraction efficiencies and matrix effects were characterized. Replicate analyses of QC material showed imprecision (CV) and bias < 10% across the measurement range. This method enables rapid, simultaneous and accurate LC/MS/MS analysis of these analytically challenging compounds, obviating the use of reversed-phase separations employing ion-pairing reagents. For Research Use Only. Not for Use in Diagnostic Procedures.
A Rapid Clinical Research Method for the Simultaneous Analysis of Urinary Catecholamines and Metanephrines by Mixed-Mode SPE and HILIC LC/MS/MS
A single extraction and analysis method has been developed for urinary catecholamines and metanephrines. Analytes were extracted using weak cation exchange SPE and analyzed using HILIC LC/MS/MS. Extraction efficiencies and matrix effects were characterized. Replicate analyses of QC material displayed excellent accuracy and precision across the measurement range. This method enables rapid, simultaneous and accurate LC/MS/MS analysis of these challenging compounds without the need for reversed-phase separations employing ion-pairing reagents. For Research Use Only. Not for Use in Diagnostic Procedures.
Plasma Metanephrines by LC-MS/MS: Method Development, Validation and Application in a Tertiary Referral Centre
A novel LC-MS/MS method for the simultaneous quantification of normetanephrine, metanephrine and 3-methoxytyramine was developed and validated. The assay displayed acceptable precision and sensitivity. Comparative sample analysis (n=215) with the LC-MS/MS method at Newcastle Upon Tyne Hospitals NHS Foundation Trust, showed a bias of +141pmol/L for normetanephrine (r2=0.97) and +26pmol/L for metanephrine (r2=0.98). The assay was consistently within the acceptable performance limits of the RCPAQAP plasma metanephrines EQA scheme for all three analytes. The assay was implemented for the diagnosis and monitoring of catecholamine-secreting tumours and retrospective review of the results revealed that the diagnostic cut-offs implemented were appropriate.
Serum 5-HIAA – a Better Marker of Neuroendocrine Tumour Than Urine 5-HIAA
We compared urine and serum 5-HIAA to assess whether serum 5-HIAA could replace urine 5-HIAA in the diagnosis and monitoring of neuroendocrine tumours (NET).
We measured 5-HIAA in 233 paired serum and urine samples from 26 healthy volunteers and 151 patients with known NET
Linear regression showed a correlation coefficient of 0.64. The sensitivity of urine 5-HIAA was 75.8%, with a specificity of 96.2%. The sensitivity of serum 5-HIAA was superior to this at 80.3%, and the test had a specificity of 100%.
We have demonstrated a strong correlation between serum and urine 5-HIAA results. The clinical sensitivity of serum 5-HIAA is superior to that of urine 5-HIAA, as is the specificity. This supports the assertion that serum 5-HIAA is a better test than urine 5-HIAA for the diagnosis and monitoring of NET.
Biological Variability of Plasma 5-HIAA
Plasma 5-hydroxyindole acetic acid (5-HIAA) measurement is used in the diagnosis and management of serotonin-secreting neuroendocrine tumours (NET). In order to calculate the reference change value of an assay it is important to know the biological variability of the analyte, so we carried out a study to determine this.
Plasma was collected from 37 post-menopausal women beyond the age of 50. Eight samples were collected over 4-8 weeks with a day interval of 6 days.
The analytical CV of the assay was 4.9%. The mean inter-individual CV was 29.47%, the mean intra-individual CV was 13.46%, with an overall variance of 32.77%. The reference change interval for plasma 5-HIAA is 23.8 nmol/L.
This will be of use when monitoring the treatment of patients with serotonin-secreting NET.
Measuring Protein Analyte Panels in Dried Blood Spots (DBS) Using an Automated SISCAPA Workflow
An automated SISCAPA method was devised using a Bravo liquid handling robot to extract proteins from dried blood spots, digest with trypsin and enrich specific proteotypic peptide analytes in 96-well plate format. This was followed by LCMS quantitation versus stable isotope labeled internal standards. A set of high-abundance proteins of plasma and red cells was measured to yield surrogate hematocrit and total plasma load and establish a normalization algorithm. Two 11-plex panels of clinically-proven cardiovascular and inflammation markers were measured from a single DBS punch across longitudinally-collected patient samples. The panel proteins span 8 orders of magnitude in abundance, with lowest present at 1-10 ng/mL range. Up to 400 patient samples were processed and analyzed in a working day.
Targeted Metabolomics and Quantitative LC-MS Analyses in the Research of Kidney Disease
Chronic kidney disease (CKD), a common disease in Australia, is characterised by an accumulation of compounds in the blood (“uraemia”) caused by lower excretion via urine due to kidney malfunction. The presented work investigates the metabolic picture of uraemia in serum and urine, using RP-LC coupled to mass spectrometry. Targeted metabolomics experiment (Thermo LTQ-Orbitrap) led to quantitative analysis of selected compounds (Waters Xevo QqQ). The results include hemodialysis clearance of uremic compounds; relationship between compounds of interest and CKD-accompanying conditions (clinical depression; premature cardiovascular disease). The importance of the tryptophan-kynurenine pathway in relation to CKD is also highlighted.
Improving Turnaround Times for Trace Element Screening of Hip Replacement Patients Using the CSols Links for LIMS System and Thermo X Series ICP-MS Spectrometer
Metal-on-Metal (MoM) hip replacement devices are used extensively to help patients with hip damage caused by arthritis or fracture. In 2012 the UK's MHRA issued an alert advising that chromium and cobalt whole blood screening should be undertaken on all patients with MoM hip replacements. Faced with a large increase in Co/Cr testing to meet the demand for screening the Supra-Regional Trace Element Assay Service laboratories in Guildford deployed CSols' Links for LIMS software to electronically report results analysed on their X Series ICP-MS spectrometers to the Clinisys' WinPath LIMS system, reducing transcription and saving analysis times.
Trace Element Levels in Korean General Population
We measured trace elements in Korean general population with inductively coupled plasma-mass spectrometry (ICP-MS). Whole blood specimens with trace metal free EDTA tube from 942 persons were collected. The concentrations of As, Cd, Hg, Pb, and Se in whole blood were analyzed. The mean concentrations of As, Cd, Hg, Pb, and Se were 8.6 ug/L, 1.0 ug/L, 4.6 ug/L, 19.2 ug/L, and 0.2 mg/L, respectively. Influences of age and sex on the values were considered. The mean levels of As, Hg, Pb, and Se were higher in male population and except Cd. The mean levels of all elements were higher in elder population. The levels presented in this study provide baseline levels in Korean general population.
A Spatially-aware Peak Picking Method for MALDI-imaging Data from TOF and FTICR Mass Analyzers
MALDI-imaging is a spatially-resolved mass spectrometric technique which can obtain the spatial distribution of hundreds of molecules in a thin tissue section. Manual analysis is tedious since it requires visual examination of all m/z-images. We introduce a novel method that automatically detects structured m/z-values without specifying a region of interest and without manual visual examination. Our parameter-free and unsupervised method provides fully-automatic selection of spatially informative m/z-images. The method also complements spectrum-wise peak picking increasing its sensitivity, as it does not depend on peak intensity, but is based on the novel measure of spatial chaos of the corresponding m/z-image.
Identification of Bruch’s Membrane Composition by Imaging Mass Spectrometry
Identification of the molecular constituents involved in Bruch’s membrane changes is necessary for understanding age-related macular degeneration (AMD). By utilizing MALDI-IMS, we determined the spatial localization of molecules present in Bruch’s membrane of an AMD patient and a control patient. We characterized regions abundant in the autofluorescent extracellular material, drusen, as well as features corresponding to lesions in the posterior pole. AMD had a specific molecular signature distinct from normal aging. These data show that imaging of Bruch’s membrane by MALDI-IMS will be a useful tool for the identification of the molecules responsible for diagnostically relevant clinical features of AMD.
Glycopathology of Aggressive Prostate Cancers Using N-Glycan MALDI Mass Spectrometry Imaging of FFPE Tissues and Biopsy Samples
A new MALDI imaging mass spectrometry workflow for simultaneous analysis of multiple N-linked glycans in standard FFPE tissue slides, tissue microarrays and biopsy cores for prostate cancers will be presented. Following antigen retrieval processing and on-tissue digestion with peptide N-glycanase, over 50 glycan species can be simultaneously detected. The approach is being applied to identify glycans that distinguish indolent from aggressive forms of prostate cancers. Methods to confirm on-tissue confirmation of glycan compositions, and efficient computational analysis workflows will also be described. The advantage of the approach, especially for heterogeneous prostate tumors, is the spatial correlation of the glycan distributions with standard pathology scoring.
UPLC-MS/MS Based Activity Assay for Determination of Mevalonate Kinase
Mevalonate kinase (MK) catalyzes the transformation of mevalonic acid to mevalonate-5-phosphate (MVAP). Mevalonate kinase deficiency is an autosomal recessive disorder, which can lead to various clinical manifestations reaching from the milder hyperimmunoglobinemia D with periodic fever syndrome to the more severe mevalonic aciduria. The disease severity correlates closely with the residual activity of the enzyme. To characterize the in vitro activity of recombinant human MK protein variants we developed an assay, which was based on the quantification of MVAP by isotope dilution UPLC-MS/MS. MVAP was detected with high accuracy (± 2.7%). The total precision of the whole assay was 8.3%.
Selective Screening for Inborn Errors of Metabolism by Tandem Mass
Spectrometry in Egyptian Children
In order to enhance awareness and promote registry for inborn errors of metabolism (IEMs) in Egypt, we aimed to evaluate the prevalence and main clinical findings of IEMs detectable by tandem mass spectrometry (MS/MS) among high risk pediatric patients presenting to our tertiary care facility at Cairo University Children's Hospital over a period of 5years .During this period 3380 Egyptian children suspected of having IEMs were analyzed by MS/MS. A relatively high number of patients (203/3380 (6%)) were confirmed with 17 different types of IEMs. The development of a nationwide screening program for IEMs is mandatory.
Evaluation of a New Commercial Complete Solution for New Born Screening and Comparisons with Established Methods
LC-MS/MS is a powerful tool for the study of metabolic disorders. The simultaneous analysis of amino acid and acylcarnitine panels can provide information on over 40 metabolic disorders such as Phenylketonuria (PKU) and Medium Chain Acyl-CoA Dehydrogenase Deficiency (MCAD). We present here a discussion of the results obtained using a new commercially available kit for the LC-MS/MS analysis of these compounds, with performance comparison and evaluation with existing commercial kits on the market today.
The Use of a New Meta-calculation Software for Automated Data Processing of Tandem MS for Inborn Error Metabolism Research
The use of Tandem MS for inborn error metabolism research started in 1990. With advancement of Tandem MS technology, more compounds can be detected and quantified using a simple sample introduction method such as flow injection with isotopic internal standards. A major challenge is to process a large quantity of generated data efficiently without transcription errors. We compared the result from a new meta-calculation software with a manual process on 100 donor samples for a total 3200 calculations. The new software significantly reduced processing time from hours to minutes and showed agreement with the manual calculations.
Evaluation of Derivatised and Non-derivatised Methods for Analysis of Amino Acids and Acylcarnitines in Dried Blood Spots Using a Novel Triple Quadrupole Mass
Flow injection tandem mass spectrometry (FIA-MS/MS) has been routinely used for analysis of amino acids and acylcarnitines in dried blood spots from newborn in order to detect inborn errors
of metabolism. We evaluated a commercially available kit and an in-house developed assay, on both derivatised and non-derivatised methods, using a novel Thermo ScientificTM TSQ EnduraTM
Triple Quadrupole MS. The exceptional sensitivity and specificity of TSQ EnduraTM MS SRM mode enabled us to reach or exceed the LOQ of amino acids and acylcarnitines well accepted in the industry, prove robust and precise method, on either derivatised or non-derivatised method.
Metabolomics Approach to the Study of Allergic Disease in Paediatric Research
Metabolomics characterized all metabolites in a biological sample. Since allergy has a high incidence in children, we apply metabolomics and mass spectrometry to the study of metabolites present in urine to highlight the possible markers. We recruited 25 children: positive (15) and negative (10) to allergy. The analysis was performed by mass spectrometry coupled to Ultra Performance Liquid Chromatography with two chromatographic columns, the samples were analysed by ESI positive and negative and the data were processed by multivariate statistical analysis; that revealed 4 variables are particularly significant of witch two were able us to predict the response to the test.
Metabolic Phenotyping of Bile Acids - Standardized Quantitative Bile Acids Analysis in Human Plasma/serum and Mouse Plasma on Different (U)HPLC-MS/MS Platforms
Bile acids are considered not only as endogenous markers for liver cell functions, but also as signaling molecules regulating triglycerides, cholesterol and glucose metabolism as well as inflammatory processes and apoptosis. Accurate determination of individual bile acids and their conjugates is very important in accessing liver damages as well as hepatic and biliary tract diseases, colon cancer, atherosclerosis and type 2 diabetes. We have developed and validated a standardized (U)HPLC-ESI-MSMS assay for the analysis of ca. 20 bile acids from only 10 µL human plasma/serum or mouse plasma samples. The panel consists of cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid, muricholic acids and their glycine as well as taurine conjugates.
Shortened Validation Procedure for a Method of Quantitative Analysis of 45 Amino Acids in Plasma on Tandem Mass Spectrometer
The validation was carried out using aTRAQ™ reagent manufactured by AB Sciex. Amino acids were quantified on API 3200 mass spectrometer. Shortened validation procedure for the method included testing of between-run and within-run imprecision, inaccuracy, limit of detection, limit of quantitation and linearity. Calculated values of the coefficient of variation (CV) for within-run and between-run imprecision and inaccuracy for most amino acids were lower than 20%, with the exception of glutamine (CV=24%). Limit of detection (LoD) and limit of quantitation (LoQ) were lower than 2 µmol/L for most amino acids except for serine, glycine and ß-alanine which had somewhat higher LoQ. The method was linear up to 1000 µmol/L for most amino acids. Based on results of shortened validation procedure, it was concluded that the method is acceptable for routine laboratory practice.
Rapid Quantification of Cortisol, Cortisone, Dexamethasone and Prednisolone in Human Saliva and Hair by Liquid Chromatography – Tandem Mass Spectrometry
Reliable determination of cortisol and cortisone concentration in saliva and hair is of interest in the assessment of stress-associated adrenocortical function. To this purpose we developed a robust method for the reliable, simultaneous quantification of glucocorticoids via LC-MS/MS, overbearing the shortcomings of immunological analysis. With excellent precision (2-7%), very good recovery rates (95-115%) and a total runtime of 4.5 min we proved the benefits of LC-MS/MS over immunoassays. Moreover, we analysed 2500 samples of the LIFE Child Depression study and demonstrated the massive impact of preanalytical factors on hair analysis, enabling proper evaluation in clinical routine diagnostics and epidemiological studies.
Metabolic Phenotyping: A New Tool Enabling Drug Response Prediction and Personalized Medicine
Metabolic phenotyping provides a surrogate marker of individual genetic disposition, somatic changes, acquired adaptations and exposition to pathogens, environment and alimentation. Allowing disease diagnosis and subclassification, treatment prediction, drug response and identification of pathophysiological processes of different diseases.
Therefore enabling an early diagnosis and prognosis of diseases yet lacking of precise diagnostic parameters.
Biocrates’ AbsoluteIDQ® Kit solutions and the in-house lab service enable the standardized, quantitative, and quality controlled analysis of over 630 relevant metabolites.
These data allow metabolic pathway analyses and identification of potential drug targets as well as their mode of action. Combined with other “omics”-techniques, metabolic phenotyping provides a system biological diagnosis for an improved personalized medicine approach.
Impact of Pre-analytical Variations in Metabolic Phenotyping
MS-based metabolic phenotyping is a powerful tool with a variety of application areas in life sciences. Blood samples are a key resource for the biomedical research, but there is no consensus on the sample type, especially the choice of anticoagulant. This study compared the concentrations of 186 endogenous metabolites in different plasma types and serum, collected from the same person at the same time, by the application of the AbsoluteIDQ p180 Kit, a MS-based quantification assay.
Our study showed that the different methodologies used for blood sampling did not affect the analytical performance. However, significant differences in the metabolomic profiles were found between the sample types. In conclusion, the sample type should be clearly defined and maintained throughout a study. And, thus, it is not recommended to compare data obtained from different samples types.
Preeclampsia Risk Stratification Early in Pregnancy: Conversion of a Promising Metabolomics Discovery into a LC-MS Based Clinical Assay
Basic metabolomics research has uncovered that combinations of blood borne metabolites can risk-stratify women early in pregnancy according to their risk of developing pre-eclampsia later in their pregnancy. Since then, a company has been established which is dedicated to translating this finding into a tool for health care providers and pregnant women. A targeted approach is being developed whereby ca. 40 metabolites are (semi-) quantified using liquid chromatography-tandem mass spectrometry. An update on the method development progress as well as an overview of the clinical studies lined-up to verify and validate the pre-eclampsia risk stratification test will be discussed.
Identification of Marinobufagenin in Plasma as a Promising LC-MS Assay for Preeclampsia Risk Assessment
Marinobufagenin (MBG) is a bufadienolide cardiac inotrope which has a growing interest in the early diagnosis of volume expansion-mediated hypertensive states such as preeclampsia.
This endogenous compound inhibits the α1 isoform of Na+,K+-ATPase, resulting in hypertension and natriuresis. The enhanced production of MBG in preeclamptic patients has been described, and demonstrated the need for a sensitive analytical method to detect MBG in plasma at low levels. Currently, only marinobufagenin-like material has been found in humans. The identification of marinobufagenin in non-pregnant human plasma utilising a LC-MS assay will be presented, leading to a promising perspective concerning the preeclampsia risk assessment.
Analysis of Isoprenoid Pathway Metabolites by LC-MS
Isoprenoid pathway metabolites such as the isoprenoidphosphates and isoprenoid-pyrophosphates are central metabolites leading to sterols, dolichols, ubiquinones, prenylated natural products and proteins. It is therefore of much interest to develop new high-performance separation methods which are able to determine the whole range of isoprenoid-
(pyro)phosphates. New HLPC-MS methods for the simultaneous analysis of the stereoisomeric dimethylallyl- and isopentenyl-(pyro)phosphates respectively have been established using the cyclo-dextrine-based stationary phase. Whereas the separation of these polar single unit isoprenoid-(pyro)-phosphates is based on selective ionic interactions on a cyclodextrin-based stationary phase, the separation of the larger isoprenoid-
(pyro)phosphates has been achieved with IPC-UHPLC-MS.
High-Throughput Metabolomics in the Epidemiological Study of Metabolic Disease
At the MRC HNR Lipid Profiling and Signalling group, we are performing high-throughput liquid chromatography and mass spectrometry analysis in large-scale epidemiological studies (>5000 samples ~ 600 per week). We aim to elucidate the involvement of metabolic pathways in the progression and diagnosis of metabolic disease through the detailed profiling of lipids and other metabolites. Currently, two epidemiological cohorts are being investigated with different chromatography and mass spectrometry approaches. While having similar aims for the elucidation of important metabolic pathways for metabolic disease, the scaling of different instrumental and data processing methods requires careful consideration of a few critical details.
LC-MS of Chiral Hydroxycarboxylic Acids
Optically active hydroxycarboxylic acids which have been related to inherited and acquired diseases are of great interest for biomedical research and applications in clinical metabolomics. LC-MS methods for the direct separation of the D- and L-enantiomers of a series of chiral 2-hydroxycarboxylic acids and 2-hydroxyglutaric acids, without the need for derivatization, have been developed. The chiral differentiation and quantification of (S)-2-hydroxyglutaric acids and (R)-2-hydroxyglutaric acids is not only important for the analysis of inborn errors of metabolism like the D-2-hydroxyglutaric aciduria type I and type II as well as L-2-hydroxyglutaric aciduria, but also for oncometabolite analysis in gliomas and glioblastomas.
Back to the Future of Human Metabolites for Central Biochemical Pathways
The understanding of central biochemical pathways and their regulation is key for molecular descriptions of human health and disease. Although central pathways are well-established milestone discoveries of classical biochemistry, many metabolites of such central pathways have not been available. As mass spectrometry analyses require pure metabolite standards, an initiative to fill these gaps has been started. New tools for the analysis of highly polar carboxylated and phosphorylated metabolites have not only proven useful for the development of new metabolite standards, but also in such widely different applications like analysing inborn errors of metabolism, cancer- and other disease-related metabolites.
Characterizing the Chemotypic Landscape of Cystic Fibrosis Sputum
In cystic fibrosis (CF) patients, mutations within the CF transmembrane conductance regulator (CFTR) lead to decreased mucociliary clearance and accumulation of thick and sticky bronchial mucus providing a nutrient rich environment for opportunistic pathogens to flourish. It is well established that microbes produce a number of organism specific molecules to interact with their environment. These specialized metabolites function as toxins, antibiotics, redox active molecules and nutrient scavenging entities. With the advent of modern mass spectrometry techniques, we can begin to unravel the hidden chemical communication in complex microbial communities. Herein, we describe the application of MS/MS based molecular networking to identify key Pseudomonas aeruginosa metabolites in clinical CF sputum samples.
Drug Monitoring of Antibiotics in Critically Ill Patients
Therapeutic drug monitoring (TDM) is well established for aminoglycosides and glycopeptides. However, there is currently no TDM for various other classes of frequently used antibiotics. Substantial variability of serum concentrations of several antibiotics with often inappropriate levels have been described in previous studies on critically ill patients. Here we present our results of a prospective observational study measuring antibiotic blood concentrations in critically ill patients with infections. The variability of blood concentrations of antibiotics was evaluated by LC-MS/MS and related to preliminary therapeutic ranges. This study should help to implement a routine TDM for different antibiotics in future.
Mass Spectral Advances in Diagnostic Microbiology: from Electrophoretic Typing to LC-MS/MS-based Approaches
SDS-PAGE combined with electrospray tandem MS (GelC-MS/MS) is frequently used for proteomic analysis. However, its use as a platform for microbial proteotyping has not been systematically investigated. Here, we subjected members of the family Enterobacteriaceae including taxonomically indistinguishable species such as E. coli and Shigella spp. to such analyses to explore the potential of this approach. By using the optimised database and proteome profiling, we could confidently identify E. coli and characterise strain-specific virulence factors, differentiate shiga-toxin negative from positive strains, and demonstrate that GelC-MS/MS has the potential to simultaneously combine strain identification with key pathogenic properties of an isolate.
Validation and Implementation of MALDI-TOF: A Quick and Easy Method for Bacterial Identification from Clinical Samples
The Department of Microbiology at Northampton General Hospital used clinical samples to evaluate the performance of the Bruker Biotyper MALDI-TOF-MS system for the routine identification of bacterial and yeast isolates from cultures compared to conventional identification methods. The results validated microbial identification using MALDI-TOF and support the replacement of existing identification methods. Because of its simplicity, speed, and accuracy, the MALDI-TOF whole cell approach promises to become a standard method of bacterial identification in clinical laboratories. Related to this, the project developed the workflow in the laboratory and looked at the turnaround time and cost associated with the implementation.
A MALDI-TOF MS and Stable Isotopes-based Approach Towards Rapid and Reliable Detection of Resistance Against Bacteriostatic Antibiotics
Recently a method based on stable isotope labelling by amino acids in cell culture was introduced for rapid detection of antibiotic resistance. Instead of visible growth the novel method measures bacterial protein biosynthesis in the presence of antibiotics. So far the method has been described to be successful for several bactericidal substances. This proof of principle study demonstrates that the method works equally well for bacteriostatic antibiotics (Linezolid, Erythromycin, Clindamycin). 49 Staphylococcus spp. isolates were analysed, where clear distinction between resistant and susceptible isolates was achieved within an incubation time of 1.5 h.
Quantitative MALDI-TOF MS for Rapid Susceptibility Testing in Septic Patients
New methods for rapid detection of antibiotic resistance are a timely topic in clinical microbiology. We evaluated the use of a novel quantitative MALDI-TOF MS approach for rapid detection of antibiotic resistances directly from patient derived blood cultures. A total of 30 consecutive blood cultures microscopically diagnosed with Gram-negative rods were tested for resistance against Cefotaxime and Ciprofloxacin. Our classification into susceptible and resistant strains was in complete accordance with the conventional method (E-test). In combination with direct identification of the pathogen the novel method could be a promising option for early therapeutic guidance in septic patients.
Evaluation of the Bruker Biotyper and VITEK MS MALDI-TOF MS systems for the identification of difficult-to-identify bacteria isolated from clinical specimens
The purpose of this investigation was to evaluate the analytical performance characteristics of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of unusual organisms. We evaluated the accuracy of two MALDI-TOF MS systems, bioMérieux VITEK MS (database v2.0) and Bruker Biotyper (software version 3.0), for the identification of the most difficult and/or unusual microorganisms isolated from clinical specimens. Our study included 174 bacterial isolates recovered from clinical cultures at Barnes-Jewish Hospital, St. Louis, MO, USA from 2009 to 2013, representing 50 genera and 52 species. We found there was no significant difference in the number of organisms identified to the genus level, species level, unidentified, or misidentified by the two MALDI-TOF MS systems.
An Evaluation of Biphenyl Chemistry to Aid in High-Throughput Bioanalytical LC-MS/MS Analyses
LC-MS/MS has become commonplace in the bioanalytical laboratory, as it can produce high throughput, high data quality analyses. The use of fully porous UHPLC and superficially porous HPLC columns are often used to increase the efficiency and peak capacity, and to decrease the analysis times. While these column advancements do impact efficiency, they do not directly impact selectivity or retention, which are the prominent parameters to analyte resolution.
We investigated a biphenyl based column chemistry to determine the effect on sample throughput and data quality in bioanalytical separations. From these applications we hope to demonstrate the advantages of the biphenyl-based stationary phase on throughput, data quality, sensitivity, and compound resolution.
LC-MS Analysis of Non-labeled Amino Acids on a Novel Mixed-mode HPLC Column
There are several established methods for analyzing amino acids, but each of these methods has disadvantages. The pre-labeled method has problems with derivitization efficiency and cost, while the post-labeled method is usually not compatible with LC-MS due to non-volatile mobile phases.
We have developed a novel amino acid separation column for LC-MS(/MS) which can analyze the complete array of 55 amino acids: 1) high throughput separation with Leu/Ile separation in 5min, and 2) simple gradient separation in 1 min or 10min. In addition, no prederivitization is required, and a standard LC-MS(/MS) system is sufficient for the analysis.
In this presentation, we will show the sensitivity and application for amino acids in serum.
High-Throughput Validated Method for the Quantification of Rufinamide in Serum Using Ultra Fast SPE-MS/MS
A fast, sensitive and specific method has been developed and validated for the quantitation of rufinamide in serum. This method utilizes an online SPE extraction using a Rapidfire 300 system and detection with an Agilent 6490 MS/MS using electrospray ionization in positive ion mode. The assay is linear from 0.5 – 60.0 µg/mL with an r2= 0.9997. The intra-day and inter-day assays (n=20) showed CVs of <8% and sample comparison samples showed good correlation demonstrated by a slope= 0.9916 and r2= 0.9739. This robust method for the quantitation of rufinamide offers cycle times of <20 seconds per sample.
Simultaneous Identification and Quantification of Triacyglycerol Species in Human Plasma by Flow Injection Electrospray Ionization Tandem Mass Spectrometry
Civilization diseases like atherosclerosis and type II diabetes are associated with elevated levels of triacylglycerol (TAG). Using conventional enzymatic methods, the fatty acid distribution in TAGs cannot be differentiated. With increasing knowledge of the effects of fatty acid distribution in TAGs, it is necessary to study the TAG molecular species. Tandem mass spectrometric detection was performed by neutral loss scans on an AB Sciex API 4000 triple quadrupole mass spectrometer with positive electrospray ionization. By coupling flow injection analysis with electrospray ionization tandem mass spectrometry, we had been able to establish a novel, reproducible and semi-quantitative method for the identification and quantification of 19 TAG molecular species in human blood plasma. Sample preparation was simple protein precipitation with toluene/methanol (1:1 v/v).
Rapid, Fully-automated Plasma Clozapine and Norclozapine Analysis Using AC Extraction Plate Technology and Isotopic Internal Calibration MS/MS
The analysis of plasma clozapine and N-desmethylclozapine (norclozapine) for therapeutic drug monitoring (TDM) purposes is well-established. Liquid-liquid extraction combined with isotopic internal calibration provides flexible and robust analytical methodology. To further develop sample processing fully-automated sample preparation using novel AC Extraction PlatesTM and a robotic liquid-handling platform (both Tecan Schweiz AG) has been adopted. Clozapine, norclozapine, and deuterated internal calibrators were simultaneously extracted from plasma samples (approximately 20 minutes/96 samples) and extracts were analysed directly using strong cation-exchange LC-MS/MS. The total LC analysis time was 4 minutes.
Single Step Separation of Plasma from Whole Blood without the Need for Centrifugation Applied to the Quantitative Analysis of Warfarin
Achieving a dried plasma sample from whole blood without the need for centrifugation creates new opportunities in blood sampling strategies for quantitative LC/MS/MS bioanalysis. Dried plasma samples were generated by gravity filtration of a whole blood sample through a laminated membrane stack allowing plasma (or what may be referred to as a non-red cell matrix) to be collected, dried, transported and analysed by LC/MS/MS. The process of gravity filtration takes 3 minutes to complete and a further 15 minutes to air-dry the 2.5uL plasma sample. The dried plasma sample disc can then be removed from the base card and stored, transported or analysed. This novel plasma separation technology was applied to the quantitative LC/MS/MS analysis of the coumarin anticoagulant, warfarin, in blood samples.
Next Generation Plasma Collection Technology for the Clinical Analysis of Temozolomide by HILIC/MS/MS
A novel approach has been developed for the quantitative determination of circulating drug concentrations using a plasma extraction technology. Plasma extraction was achieved by applying a blood sample to a laminated membrane stack which allowed plasma (or non-red cell matrix) to flow through the asymmetric filter whilst retaining the cellular components of the blood sample. This technology has the potential for a simplified clinical sample collection including the finger prick approach. Plasma separation card technology was applied to the quantitative analysis of temozolomide (an oral imidazotetrazine alkylating agent used for the treatment of Grade IV astrocytoma, an aggressive form of brain tumour) in blood samples by HILIC/MS/MS.
Enhanced Quantitation of Structurally Similar Proteins Using a Novel Acquisition Protocol and Mass Spectrometric Analysis
Metabolism of drugs by the Cytochrome P450 superfamily is pivotal in determining their disposition, safety and efficacy. Since drugs may induce expression of several isoforms of Cytochrome P450, they may enhance their own turnover, increasing the risk of toxic metabolite formation or adverse interactions with co-ingested compounds. Thus P450 profiling is a fundamental aspect of drug safety evaluation. The Cytochromes P450 share extensive structural similarity, so that antibodies are incapable of discriminating every isoform, plus mRNA levels do not correlate well with protein. SWATHTM is a data-independent MS method for label-free quantification which enables closely-related proteins to be quantified retrospectively through post-acquisition extraction of specific peptide ions, and is thus perfectly suited to P450 profiling.
An Innovative Variable-energy Electron Ionisation Technology Applied to GC-TOF MS Metabolomics Applications
Traditionally, GC-MS instrumentation is operated at a single electron energy of 70 eV to maximise ionisation efficiency. However, this often results in significant fragmentation and loss of the molecular ion signal. The presence of the molecular ion can substantially increase the confidence in metabolite identification. Recently, a new and innovative variable-energy electron ionisation source, Select-eV, has been marketed by Markes International to allow data acquisition at both high (70eV) and low (<15eV) energies with no loss in sensitivity. The presentation will compare GC-MS data acquired at energies of 14eV and 70eV and assess the applicability of applying this novel methodology for the study of mammalian biofluid metabolomes.
Comparison of LDTD with Traditional LC-MS for Quantitative Screening of Benzodiazepines in Urine Using Benchtop Quadrupole Orbitrap Mass Spectrometer
Laser diode thermal desorption coupled with high-resolution accurate-mass spectrometry was evaluated to support forensic toxicology screening of benzodiazepines in urine and was compared to traditional LC-MS approach. Samples were processed by hydrolysis and liquidliquid
extraction. All compounds were linear from 1000 ng/mL to 1 to 10 ng/mL using LDTDMS.
Results from LDTD compared to LC-MS were good with correlations >0.9. QCs had accuracies and precisions within 30%. Internal standards in donor samples show limited and acceptable matrix effects. LDTD coupled to HRAM mass spectrometer offers semi-quantitative
screening solution making it an attractive choice for forensic toxicology laboratories seeking high throughput.
Confirmatory Determination of Buprenorphine and Norbuprenorphine in Urine Using a High-Throughput LC-HRAM-MS/MS Methodology
Forensic toxicology labs monitor levels of buprenorphine and its major active metabolite,
norbuprenorphine, in urine in order to determine compliance with treatment for chronic
pain or opioid addiction. LC-MS/MS is currently the most popular method used for confirmation. Here we demonstrate that the use of a two-channel UHPLC system with a high resolution accurate mass MS equipped with segmented quadrupole allowed for highest specificity, sensitivity, and sample throughput as compared to conventional LCMS/
MS. This methodology resulted in achieving a sample cycle time of 1.07 minutes and dynamic linearity range of 5 – 2000ng/mL with correlation coefficients > 0.996 for both analytes.
Analysis of Pain Panel Medications in Urine on Raptor™ Biphenyl by LC-MS/MS
Restek Corporation, 110 Benner Circle, Bellefonte, PA 16823
For nearly a decade, the Restek™ Biphenyl has been the column of choice for clinical diagnostic and Pain Management drug screening testing because of its ability to provide highly retentive, selective, and rugged reversed-phase separations of drugs and metabolites. By bringing the speed of Superficially Porous Particles to the Biphenyl family, Restek’s Raptor™ Biphenyl provides clinical labs with an even faster option for a wide variety of clinical assays. Drug Screening applications can be difficult to optimize and reproduce due to the limited selectivity of C18 and phenyl-hexyl phases. Using Raptor™ Biphenyl columns pain management analysis can be performed with a 5-minute cycle time and complete isobaric resolution. Popular competitor methods have tailing peaks, longer run times, and co-elutions.
Proteomic and Pharmacologic Characterization of Moroccan Cobra Venom
Moroccan cobra Naja haje legionis is one of the most dangerous snakes implicated in the pathogenesis of snakebites in Morocco. To identify the essential components of the venom involved in the pathophysiology of envenomation by the cobra Naja haje legionis, we have initially performed a proteomic characterization of the crude venom by using a combination of chromatographic techniques, tandem mass spectrometry and interrogation of the Swiss-Prot database. Secondly, the pharmacological properties of the venom of Naja haje legionis were also studied by using tissue preparations from rodents and chicks.
Kappa and Lambda Light Chain Molecular Mass Distribution Using Mass Spectrometry
Mass spectrometry (MS) can be used to characterize intact immunoglobulin light chains (ILC). In this study we qualitatively and quantitatively evaluated serum kappa and lambda total light chains in normo-, hypo- and hyper-gamma patients using immunonephelometry and MS on an ABSciex-TripleTOF 5600 (microLC-ESI-Q-TOF-MS). A pre-analytical purification step for IgG using Melon Gel (Thermo Scientific) was used for the MS method, and IgG recovery estimated. Nephelometric quantitation and microLC-ESI-Q-TOF-MS calculation of the kappa/lambda ratio were compared yielded an equation y=1.4x+1.37 and R2 of 0.64. The results encourage us to pursue MS approaches to detect and quantitate ILC abnormalities in the study of monoclonal gammopathies, polyclonal light chains elevation in autoimmune diseases, immunodeficiencies, and post-vaccination.
Towards Clinically Actionable Quantification of Proteins by Mass Spectrometry: A Critical Appraisal of Bias and Imprecision for Serum Apolipoproteins A-I and B
Test results in medical laboratories should be traceable to standards of higher order, and within predefined criteria for analytical quality. In this study, we describe a thorough evaluation of bias and imprecision, according to CLSI-guidelines EP15 and EP9, for mass spectrometry-based quantification of serum apolipoprotein A-I (Apo A-I) and apolipoprotein B (Apo B). The results provide (1) a systematic overview of the impact of trypsin digestion, calibration, internal standardization, peptide selection, LC-MS/MS performance, and matrix-effects on analytical performance, and (2) a proof-of-concept to achieve metrologically traceable results within the minimal total allowable error based on biological variation.
Standardized Targeted Proteomics Approach for the Simultaneous Determination of Eight Apolipoproteins in the Leipzig Heart Study
Sample preparation strategies for absolute quantification of eight apolipoproteins including Apo E and J in human plasma by microLC-MS/MS using proteotypic tryptic peptides and corresponding isotope labeled peptides as internal standards were investigated to establish a standardized sample pretreatment protocol for high-throughput analysis using only 3 µL of sample material. Method validation revealed adequate lower limits of quantification as well as coefficients of variation < 13%. Method comparison with commercial immunoassays for Apo A-I and Apo B-100 in 1000 samples of the Leipzig Heart study showed good agreement. Consequently, the distribution of eight apolipoproteins in cardiovascular disease was reliably determined.
Plasma Proteome Profiling of Atherosclerotic Disease Manifestations Reveals Elevated Levels of the Cytoskeletal Protein Vinculin
Atherosclerosis is a chronic disease of the arterial wall, recognized as the leading cause of mortality and morbidity.
Here we used quantitative proteomics to identify proteins with altered levels in plasma from four patient groups: 1) no coronary calcium, 2) high coronary calcium, 3) stable atherosclerotic disease, and 4) acute coronary syndrome (ACS). The top-most elevated protein, vinculin (Vcl) displayed an increasing expression profile and verification by immunoassays and multiplex SRM-MS assay confirmed significant up-regulation of Vcl in the ACS group (P<0.004) along with known markers of atherosclerosis. Further independent validation in ACS subgroups of myocardial infarction confirmed these findings.
Quantitative Phosphoproteomics Analysis of Malignant Ovarian Tumors in Mice by LC-MS and Labelling with Tandem Mass Tags
Ovarian cancer ranks the fifth in cancer deaths among women. Mass spectrometer based phosphoproteomics analysis can provide important insight in understanding the molecular mechanism in this disease development process. In our study, C57BL/6 mice were challenged with MOSE IG-10 cell line for producing later stage ovarian cancer. The malignant tumors were then lysed, reduced, alkylated, digested, and labeled with 6-plex tandem mass tag reagents. The labelled phosphopeptides were enriched with TiO2 column and were analyzed on a LTQ-Orbitrap mass spectrometer coupled with a nano flow HPLC. We identified several proteins with enhanced phosphorylation activity in malign ovarian cancer.
Absolute and Multiplex Quantification of Therapeutic Monoclonal Antibodies in Serum Samples by LC-SRM Using PSAQ Standards
Since the first approval of a therapeutic monoclonal antibody drug in the late nineties, over 400 therapeutic monoclonal antibodies (mAbs) were undergoing in preclinical development or clinical evaluation. Due to high specificity and efficacy, therapeutic mAbs have become a major class of therapeutic compounds for the treatment of cancers, infectious diseases, allergies, inflammation, and auto-immune diseases. To date, immunoassays, such as ELISA, remain the most sensitive, specific and selective technologies used for quantifying mAbs in biological fluids. However, it requires specific developments for each mAb, suffers from matrix-effect and is species-dependant, what is time-consuming, costly and not really suited for early phases of developments. Thus, the development of innovative and fit-for-purpose MS-based quantification methods represents a challenge for pharmacokinetics studies.
Quantitation of Multiplexed Serum Apolipoproteins by Stable Isotope Dilution- Multiple Reaction Monitoring-LC-MS/MS
An LC-MS/MS method was developed for simultaneous measurement of six serum apolipoproteins: apo A-I, B100, C-I, C-II, C-III, and E. The analytical performance was evaluated using CLSI EP15 and EP9 protocols, demonstrating a total CVa below 15 % for all twelve peptides. In addition, LC-MS/MS results correlated well with CE-marked immunoassays for quantitation of serum apo A-I and apo B100 (R > 0.975) in 100 normo- and hypertriglyceridemic sera. No interference was found in hypertriglyceridemic sera as compared to normotriglyceridemic sera. The laboratory-developed test has potential to improve cardiovascular disease risk stratification and treatment monitoring.
Developments Towards High-throughput Plasma Proteomics Platform for the Clinic
Fast and in-depth proteomics of blood samples has been a long-standing but unachieved goal. Here we apply a novel sample-preparation pipeline in combination with high-throughput LC-MS measurement technologies to identify and accurately quantify important clinical in short mass spectrometric measurements. Preliminary results demonstrate a depth of more than 600 proteins in less than 2 h of LC-MS time. Very short gradients (10 min) still enabled measurements of more than 200 proteins, including 42 FDA-approved biomarkers. These developments contribute to opening plasma proteomics up for routine, quantitative analysis of patient samples in clinical settings.
Studying the Effect of Natural Genetic Variation on Protein Abundance in C. elegans
Complex diseases (eg cancer) are caused by a combination of genetic and environmental factors, including lifestyle. Many of the signaling pathways involved in these diseases are conserved among species and also present in Caenorhabditis elegans a microscopic nematode. To study the influence of natural genetic variation on the abundance of proteins involved in signaling pathways, we used selected reaction monitoring and quantitative trait loci mapping. We analyzed recombinant inbred lines generated from the two genetically divergent C. elegans wild-type strains Bristol N2 and Hawaii CB4856. Our data suggest that protein levels are under strong evolutionary control than transcript levels.
Performance Evaluation of the Thermo Fisher Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer for High-throughput Top-down Microbial Proteomics
By using the Q Exactive HF mass spectrometer in conjunction with a new charge assignment and protein deconvolution algorithm, we demonstrate improved performance in protein identification in a series of high-throughput top-down proteomics experiments with complex E. coli extract.
Application of Multiple Protease Digestion with Shotgun Protein Sequencing De novo Approach to the Characterization of Hemoglobin Variants
Hemoglobin variants may be related to pathologies. To characterize hemoglobin variants, we digested hemoglobin with trypsin, lysC, and wild type and mutant alpha lytic proteases, acquired data-dependent LC-MS/MS spectra on an AB SCIEX TripleTOF 5600, and subjected the data to shotgun protein sequencing (SPS) with meta-contig assembly. As proof of concept, the analysis of human hemoglobin samples consistent with HbAE and HbAS/HbSS included contigs that contained the E26K and the E6V mutations, corroborating the HPLC results for samples consistent with hemoglobin E and S trait, respectively. Overall coverage was 90.8% for hemoglobin alpha chain and 94.6% for beta.
LC-MS Candidate Reference Methods for the Harmonisation of Parathyroid Hormone (PTH) Measurement: A Review of Recent Developments and Future Considerations
The analysis of intact PTH (PTH1-84) is important in the diagnosis of hyperparathyroidism, and in the clinical management of bone mineral disorders. Recently, LC-MS methods have been developed for quantitation of PTH1-84 using tryptic peptides, but these methods are subject to interferences due to the presence of modified (oxidised/phosphorylated) PTH species, which accumulate in patient samples. The tryptic digestion process produces non-specific tryptic peptides which cannot be used as surrogates for PTH1-84. Further work, including the use of high-resolution MS, to allow the analysis of PTH without protease digestion is required before these approaches can be considered as reference methods.
Estradiol - Investigation of Two Sample Preparation Procedures for Quantitative Analysis by LC-MS/MS
Sample preparation of Estradiol (E2) for LC-MS/MS analysis was investigated using the AC Extraction Plate™ featuring TICE™ (Tecan Immobilized Coating Extraction) technology in comparison to a Liquid-Liquid-(LL)-extraction procedure. Using the AC Extraction Plate workflow quantifiable signals for E2 were obtained down to levels around 500pmol/l of E2 on an ABSciex 4000 LC-MS/MS system while less serum was required compared to LL-extraction. For these concentrations the workflow offers the possibility of complete automation with walk-away capability. For levels below 500pmol/l of E2 an additional evaporation/reconstitution step was added to enhance signal intensity.
Targeted Quantitation of Insulin and Its Therapeutic Analogs
Detection and quantification of insulin and its analogs has become paramount for medical and athletic doping. Traditional assays lack the ability to differentiate insulin and insulin analogs due to the lack of selectivity. Therefore, a Mass Spectrometric Immnoassay was developed. Capitalizing on conserved sequences, a single antibody is used to simultaneously enrich for insulin and insulin therapeutics, while LC/MS enables differentiation of the sequence variances. A robust clinical research assay able to concurrently measure multiple insulin analogs, providing a 15 pM lower limit-of-quantification and a dynamic range of 15-960 pM, is demonstrated.
Sample Preparation Using Supported Liquid Extraction for Clinical Applications Prior to LC-MS/MS Analysis
This presentation is designed to give an introduction to the use of supported liquid extraction in a clinical environment prior to LC-MS/MS analysis. Extract cleanliness is a major factor when investigating sample preparation strategies. Data will be presented with respect to serum/plasma in terms of endogenous matrix components; specifically proteins and phospholipids. pH control and extraction solvent selection can significantly influence extract cleanliness. We will then briefly discuss three applications where extraction optimization has been performed; corticosteroids, vitamin D and steroid hormones.
The Utilization of In Vivo and In Situ Solid Phase Microextraction in Clinical Analysis
Solid phase microextraction (SPME) was used as a novel approach for in vivo and in situ extraction of drugs and endogenous metabolites from lung during in vivo and ex vivo lung perfusion (IVLP and EVLP, respectively). The concentration profile of the chemotherapeutic and its distribution in the organ as well as changes of global metabolome were analyzed with the used of LC-MS(/MS) platforms. Due to the minimum invasiveness and easy direct coupling of SPME probe with mass spectrometer the method shows great potential to be used on-site as rapid diagnostic tool.
A New, Miniaturized Method for Extracting Plasma from a Blood Drop as Part of Preparing Proteins and Metabolites for Mass Spectral Analysis
This paper describes a miniaturized membrane filtration system for removing cells and extracting plasma from a blood drop for MS analysis; by capillary action. A 2.5 uL aliquot of plasma was collected on a 6.3 mm, internal standard loaded paper disc within ~3 min; independent of blood volume or hematocrit. The plasma disc was subsequently removed and air dried; leaving a dried plasma spot (DPS). Recovery and quantification were validated in MS based vitamin D, drug, and protein assays. Using immobilized trypsin and antibodies in the DPS, analyte peptides were prepared and selected from plasma in-situ during transport; allowing MS analysis on sample arrival.
Development of SPME-LC-MS/MS Method for Concomitant Extraction of Rocuronium Bromide and Tranexamic Acid from Plasma
The presented work shows new method for determination of rocuronium bromide and tranexamic acid. The major challenges related to the analytical procedure are different properties of the drugs, their expected concentrations and instability of rocuronium in plasma samples. The proposed protocol is based on thin film solid phase microextraction followed by LC-MS/MS analysis. The developed approach offers high throughput of sample preparation and very good sample clean up thus improving time and quality of the data. Simplicity of the method and potential of coupling SPME device directly with mass spectrometer make the method applicable for onsite use in clinical facilities.
A Novel Online Cleanup Valve Solution for Quantitative Analysis of Testosterone in Serum Utilizing LC-MS/MS
Determination of testosterone levels in serum is an important measurement in clinical research. Liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS) is an essential tool in the analytical laboratories due to its high sensitivity and specificity, excellent reproducibility, and it can be used in simultaneous analysis of multiple analytes. This work describes a novel integrated valve and liquid metering system used for on-line sample cleanup coupled with a MS/MS.
High-throughput LC-MS/MS Analysis of Immunosuppressants in Whole Blood Using On-line SPE and Automated Sample Preparation
For accurate, sensitive and fast therapeutic drug monitoring (TDM) of immunosuppressants in clinical routine analysis, RECIPE has developed its ClinMass® LC-MS/MS Complete Kit, advanced - Immunosuppressants in Whole Blood (MS1100). High-throughput LC-MS/MS analysis can be realized using liquid handling systems and two-dimensional LC-MS/MS. The method shows outstanding validation results for all analytes. Close agreement of results was found for clinical samples which were processed applying automated and manual sample preparation. High throughput TDM enables preparation of 96 samples <45 minutes. Full automation is realized which significantly increases sample traceability and process security very well suited for clinical routine analysis.
High-throughput LC-MS/MS Analysis of 25-OH-Vitamine D2/D3 in Serum Using On-line SPE and Automated Sample Preparation
The determination of 25-OH-Vitamin D is widely used for the assessment of the vitamin D status, which is important for many chronic conditions and latency diseases. For high throughput analysis RECIPE has developed an automated ClinMass® LC-MS/MS Complete Kit MS7000 - 25-OH-Vitamin D2/D3 in Plasma and Serum working with liquid handling systems and two-dimensional LC-MS/MS. The fully CE IVD certified kit shows outstanding validation results. A comparison between automation and manual sample preparation shows close agreement of the concentrations of the analytes for both variants. Full automation increases significantly sample traceability and process security suiting very well into clinical routine analysis.
iST: Sample Preparation for High Throughput Clinical Proteomics
Rapid and robust workflows are crucial for day-to-day clinical applications. Especially sample preparation procedures are time consuming and limit the overall technical reproducibility of MS-based proteomics. Here we present the in-StageTip (iST) method for streamlined sample processing of complete proteomes. This simplified 3-step procedure is performed in a single, enclosed volume and allows peptide pre-fractionation in a high-throughput fashion. Applying the procedure to the cancer cell line HeLa allowed us to estimate copy-numbers of 9,667 proteins with excellent reproducibility (R2 = 0.97) in quadruplicates measurements. The in-StageTip method allows high-throughput applications with near complete proteomic coverage of highly complex samples.
Analysis of Serum Testosterone and Androstenedione for Clinical Research Using Either Manual Or Automated Extraction
Here we evaluate a UPLC/MS/MS method for the measurement of serum testosterone and androstenedione enabling investigation of metabolic dysfunction for clinical research. An analytically selective method was developed using a mixed-mode Solid Phase Extraction (SPE) sorbent in 96-well plate format. Either manual or automated extraction was employed, providing flexibility in sample preparation options. Analysis was performed using an ACQUITY UPLC® I-Class system, samples were injected onto a 2.1 x 50 mm Waters ACQUITY UPLC HSS C18 SB column using a water/methanol/ammonium acetate gradient and quantified with a Waters Xevo® TQD mass spectrometer.
For Research Use Only, Not for Use in Diagnostic Procedures.
Improved Sample Preparation and HPLC-MS Analysis of Vitamin D Metabolites from Human Plasma
The analysis of Vitamin D metabolites has continued to be a topic of interest in current research, because those serve as biomarkers for possible disease states and for vitamin sufficiency. Vitamin D is present in two forms, vitamin D2 and D3.
In this work, a LC-MS method for the analysis of Vitamin D metabolites was developed to include dihydroxy metabolites along with the epi-homologs. Special focus of this development was the chromatographic resolution of the isobaric compounds. In addition, sample preparation techniques are evaluated to reduce the impact of biological matrix ionization effects.
A Rapid Clean-up Procedure for Monitoring the Biomarker of Dimethylformamide in Hemoglobin by LC-MS/MS
This paper described a method for monitoring 3-Methyl-5-Isopropylhydantoin (MVH) , a biomarker of the degration of dimethylformamide (DMF) in hemoglobin. Cleanert MAS-B Plate with 96-well format was used for rapid clean-up of the samples. 3-Methy-5-isobutylhydantion (MIH) was applied as internal standard to quantify MVH in hemoglobin. A clean fraction was obtained for high throughput monitoring of MVH by LC-MS/MS without further concentration. Recoveries for MVH were range from 100.4% to 102.5% with RSDs were £¼4.6%. LOD of MVH and MIH were 1.0ng/mL. The study provided a solution for assessing cumulative exposure to DMF.
Comparison Between Different Process Methods of Arachidonic Acid in Plasma
This poster was a comparative study 3 different sample pretreatment processes to extract Arachidonic Acid (AA) from plasma, which involved 96-well protein precipitation plate, Brand W 96-well plate and Cleanert MAS-M 96-well plate. Protein precipitation method enjoyed a convenience due to its minimum procedures, but its recoveries of AA were 129.32%~149.02%, implying the worst purification effect which caused the matrix enhancement on mass spectrum. The recoveries of AA on Brand W 96-well plate were 5.45%~70.15%, while the recoveries of Cleanert MAS-M 96-well plate were 99.19%~106.38 which ensured an extraction procedure without reconstitution to support a rapid, high throughput assay of AA in plasma.
A Study of Stability, Robustness and Time Efficiency of a New HPLC and a New Tandem MS
Stability, robustness and time efficiency of HPLC-tandem MS are three major challenges in laboratories where a large number of samples are analyzed routinely. Here we report a study of a new design of two channels HPLC and a new design of tandem mass spectrometer to address these three challenges. A total of 1241 crashed synthetic serum samples spiked with alprazolam and isotopic internal standard were analyzed continuously in 60 hours, with additional 25 QC samples inserted. On-line turboflow column help removing sample matrix. RSD% of retention time and concentration was observed at 0.94%, 2.02%, respectively.
Highly Automated, High Precision Tryptic Digestion and SISCAPA-MS Quantification of Human Plasma Proteins Using the Agilent Bravo Platform
We report here the development of a highly reproducible, automated “addition only” method for tryptic digestion of plasma, followed by bead-based SISCAPA enrichment of target proteotypic peptides, driven through a simple software user interface. The combined protocols, taking as little as four hours total, were coupled with high throughput LC-MRM for multiplexed quantitation of plasma proteins over a wide range of abundance. The complete workflow was demonstrated for suitability for routine quantitation of target plasma proteins based upon sensitivity, multiplexing capability, scalability, reproducibility, and throughput. These characteristics make the workflow an appropriate platform for routine measurement of clinically relevant proteins.
A Simple High-throughput SPE Method to Support the Biomonitoring of Phthalate Metabolites in Human Urine Prior to LC-MS/MS
An SPE-LC-MS/MS method to quantify nine phthalate metabolites in human urine was developed. To facilitate the sample preparation of glucuronidase-treated urine samples prior to LC-MS/MS analysis, three sorbent chemistries (ISOLUTE ENV+, ISOLUTE Myco, EVOLUTE ABN) were evaluated. We found ISOLUTE ENV+ to be most suitable for the efficient and reproducible recovery of phthalate metabolites in human urine. The run time for 40 samples and 8 calibrators was 30 minutes. The optimized method allowed for LODs in the range of 0.05-0.2 ng/mL with at least four orders of magnitude in linear dynamic range for all analytes.
Evaluation of Supported Liquid Extraction for Vitamin D: 25-hydroxy and 1,á,25-dihydroxyvitamin D2/D3, PTAD Derivatization and Analysis Using UPLC-MS/MS
Vitamin D analysis has important clinical relevance with levels needing to be measured for a wide variety of reasons. This poster demonstrates a simple supported liquid extraction protocol, derivatization with PTAD and subsequent detection of both the traditional hydroxy metabolites and the biologically active dihydroxy metabolites in serum. Extracts were analysed using a Waters ACQUITY UPLC system coupled to a Quattro PREMIER XE triple quadrupole mass spectrometer. Positive ions were acquired using ESI operated in the positive MRM mode. Method performance demonstrated high analyte recoveries and low ion suppression, allowing a quantifiable range matching levels therapeutically expected for each metabolite.
Comparison of SPE Approaches for the Extraction of Thyroid Hormones: T3, RT3 and T4 Prior to LC-MS/MS Analysis
Thyroid hormones are extremely important physiologically and involved in many biological processes such as growth and development. Reverse T3 is only produced at low levels compared to T3 and most notably T4 so it is important to have a robust assay capable of detecting these low levels. This poster evaluates the performance of various resin-based ion exchange SPE chemistries for the extraction of thyroid hormones prior to LC-MS/MS analysis. Method performance will be investigated from an analyte recovery perspective with particular emphasis on extract cleanliness.
Automating an Assay for 25-OH Vitamin D in a Clinical Research Laboratory to Reduce Training Requirements for Mass Spectrometry
The analysis of 25-OH Vitamin D within the clinical research laboratory is a high throughput assay. As a consequence of the high number of samples, automation and simplification of assay is an attractive proposal to minimise sources of error and staff training requirements, reducing the need to train staff in additional Mass Spectrometry and sample preparation theories. In addition, automated handling of sample and result data significantly reduces errors associated with manual transcription. We propose here an elegant and cost effective solution for this application.
TICE – an Innovative, Easy-to-automate Extraction Technique for Small Molecule Analytes from Biological Fluids for LC-MS/MS Analysis
Tecan Immobilized Coating Extraction™ (TICE™) – a sample preparation technique for LC-MS/MS analysis – uses a simple to automate plate format for analyte extraction from biological fluids. This extraction device was applied for extraction of various 25-hydroxyvitamin D (25OHD) metabolites from serum at a robotic liquid handling system. A LC-MS/MS method was developed for quantitation. The AC Extraction Plate™ was found to be suitable for extraction of 25OHD3/2, their epimeric forms and the metabolite 24R,25-(OH)2D3. Within a set of 47 human serum samples significant amounts of 3-epi-25OHD3 were detected in infants. TICE™ enables automated extraction of various 25OHD metabolites with different polarity.
Comprehensive Study of SLE as a Sample
Preparation Tool for Bioanalysis
A simple,fast,efficient and high through-put sample preparation method is necessary to remove the interferences from plasma samples prior to LC-MS/MS in bioanalysis. This paper describes a comparison study of four sample preparation methods, LLE, PPT, RP-SPE and SLE, with spiked plasma. Dexamethasone is used in the study.The results showed that the responses of SLE are about five times higher than that of others.
Keywords: SLE plate, sample preparation, plasma.
A Fast and Effective Approach for the Analysis of Urinary Cortisol, Cortisone, Prednisolone and Prednisone Using SPE and LC-MS/MS
The existing methods for the quantification of cortisol, cortisone, prednisolone and prednisone are very diverse. While liquid-liquid extraction, protein precipitation and “dilute and shoot” procedures offer quick and dirty methodologies, these same methodologies risk increases in instrument down time and analytical column costs. We evaluated a variety of silica-based and polymer-based SPE sorbents, each of which provides a different retention mechanism, including; hydrophobic, anion-exchange, pi-pi interactions, and any combination thereof. The evaluation showed that a modified polymeric sorbent, with a unique elution solvent has been found to be a robust, reproducible and cost effective solution for the laboratory, while providing a LLOQ of 10 ng/mL in human urine, for all four corticosteroids.
Development of a LC-MS/MS-Method for the Determination of Colistin and Colistin Methanesulfonate and Application to Plasma-Samples of Critically Ill Patients
A LC-MS/MS method was developed for the determination of colistin and colistin methanesulfonate (CMS) in plasma samples of critically ill patients. More than 800 patient-samples have been analyzed. The sample preparation includes the extraction and protein precipitation with TFA/isopropanol using polymyxin B as internal standard. CMS was determined by hydrolysis prior to the determination of total colistin. The analysis was performed using ESI in positive mode. Special problems concerning standardization, stability and extraction yield will be addressed. PK-Data are derived from the measurement of patient samples under renal replacement therapy and will be compared with already published results.
A Dutch Study into the Analysis of Vitamin B6 in Whole Blood
In this study, different commercial and homemade HPLC-methods for measuring vitamin B6 in whole blood were compared with a homemade LC-MS/MS method using different sample types. The comparison of whole blood showed a proportional bias of 40% for the Chromsystems method when compared with the LC-MS/MS method whereas the INstruchemie and the homemade HPLC methods showed no bias towards the LC-MS/MS method. Surprisingly, the comparison of lyophilized blood showed no bias between methods. This lack in standardization or harmonization between methods is hampered by the absence of a reference technique or reference material for measuring vitamin B6 in whole blood.
Fast and Robust LC-MS/MS Method for Determination of the Alcohol Biomarker Phosphatidylethanol (PEth) in Whole Blood Using Automated Extraction
Phosphatidylethanol (PEth) is a direct alcohol biomarker with higher sensitivity and specificity than other indirect alcohol biomarkers used, e.g. GGT, CDT. There is also a correlation between reported alcohol intake and PEth-concentration in the blood. PEth is formed in the cell membrane of the erythrocytes only when ethanol is present in the blood.
LC-MS/MS is a necessity because of the many forms of PEth, with similar retention time and mass, present. The most abundant form, PEth 16:0/18:1, was used for quantitation.
This poster presents the whole method, from sample preparation to LC-MS/MS detection together with the validation results.
Benefits of the Orbitrap Technology in LCMS for Therapeutic Monitoring of Baclofen and Its Metabolites in Plasma and Urine Samples.
Baclofen is a centrally acting muscle relaxant, prescribed in France for the treatment of cerebral spasticity. It has recently attracted considerable attention as a potential medication for alcoholism. Variability in efficient dose has been observed in patients. Therefore it is of interest to monitor baclofen and its major metabolites in plasma. We describe a method to semi-quantify baclofen’s metabolites in a single run by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Tests were initially carried out on samples collected from patients treated in context of alcohol withdrawal and/or in cases of overdose for forensic toxicology purposes.
Therapeutic Drug Monitoring (TDM) of Various Types of Antibiotics by a Single Multi-purpose LC-MS Method
LC-MS is the method of choice for therapeutic drug monitoring of a variety of drugs, including antibiotics. Despite methods for the search of a number of antibiotics in food matrices are present in the literature, no LC-MS method is published for multi-drug quantitation of high impact antibiotics in the blood of patients treated with these drugs. We developed, validated and applied to clinical research cases an original, high throughput LC-MS method for the simultaneous analysis of cephalosporins, carbapenems, aminoglycosides and other classes of antibiotics in blood plasma of patients. The method has currently been put in clinical use for the TDM of these drugs in various clinical settings in hospitals of our region. It is apparently providing clinicians with a helpful tool for the management of severely ill patients suffering infections by various types of drug-resistant life-threatening germs.
Online Analysis of 25-OH-vitamin D2/D3 in Plasma and Serum with the Evoq Triple Quad
A robust and reliable research method to quantitate 25-OH-vitamin D2 and D3 in plasma and serum samples using the Bruker Advance UHPLC with OLE coupled to the EVOQ Elite triple quad is demonstrated. The integrated online extraction option of the UHPLC together with the ClinMass® LC-MS/MS complete kit (MS7000) provides fast and easy sample cleanup. Interlacing the online extraction and chromatographic separation reduces the overall run time to 3 minutes per sample. The calibrations showed excellent linearity with r²≥0.999. The assay had a very good interday and intraday precision with RSD <6.5% as well as high accuracy with bias < ±6%.
Maximizing Triple Quadrupole Mass Spectrometry Productivity Through the Automated Use of an Expanded Dual-Channel HPLC System with Online Sample Cleanup
This work explores increasing mass spectrometer productivity through the automated use of an expanded dual channel high performance liquid chromatography system. The complete, integrated LC/MS/MS system is comprised of a triple quadrupole mass spectrometer coupled to a configurable HPLC system, all controlled by a single software application. A previously developed method for the analysis of 25-OH vitamin D has been used for testing the capabilities of this new instrument. By staggering injections on parallel streams and switching between the two streams at the appropriate time, throughput of the integrated expanded system can double the throughput of the standard method.
Ultra-high Throughput Quantitation of Immunosuppressants in Human Whole Blood
The purpose of this study was to evaluate the usability of the Agilent RapidFire-MS/MS system using solid phase extraction (SPE) cartridges instead of chromatography columns for the quantitative analysis of cyclosporine A, everolimus, sirolimus and tacrolimus in a clinical research laboratory. Results of more than 1100 samples were compared between LC-MS/MS system and the RapidFire 360 system coupled to an Agilent 6460 QQQ mass spectrometer. Sample preparation and measurement were performed using a commercial kit, imprecision and accuracy were investigated analyzing three concentration levels of commercial QC samples.
Increased Throughput for the Analysis of Delta-9-THC in Oral Fluids Using Triple Quadrupole Mass Spectrometry Coupled with an Automated Dual-Channel HPLC System
In this work we demonstrate increased mass spectrometer productivity through the automated use of a dual channel high performance liquid chromatography system. The integrated LC-MS/MS system is comprised of a triple quadrupole mass spectrometer coupled to a configurable dual HPLC system, all controlled by a single software. The analysis of delta-9-THC from oral fluids was used to demonstrate capabilities of this system. The new HPLC system mirrors certain components of this single stream system to provide a second stream, operating in parallel to the first stream. By staggering injections on parallel streams, throughput can double as compared to standard method.
Determination of Urinary Ethyl Glucuronide and Ethyl Sulfate by LC/MS/MS for Clinical Research
Liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS) is ideally suited for the rapid analysis of multiple analytes. A highly sensitive and specific LC/MS/MS method has been developed for the quantitation of ethyl glucuronide and ethyl sulfate. A dilution procedure and a solid phase extraction (SPE) procedure are evaluated and compared based on ease of use, analyte recovery and post-extraction cleanliness. The described method achieves the required sensitivity and is capable of quantitating analytes over a wide dynamic range. Excellent reproducibility was observed for all compounds (CV < 5%). All calibration curves displayed linearity with an R2 > 0.995.
The Separation and Analysis of Opiates and their Glucuronide Metabolites in Urine Matrix Negating the Need for Hydrolysis During Sample Preparation
Opiates and their metabolites can be challenging to analyze using LC/MS due to a relatively large number of their analytes having the identical empirical mass and subsequent similar fragmentation patterns in MS/MS. The need for good chromatographic separation is therefore paramount as is the need to reduce complicated sample preparation techniques. Opiate glucuronides pose the greater separation challenge in reverse phase chromatography due to their
highly polar nature and are, therefore, routinely hydrolyzed during sample preparation, a process that removes any sugar group returning the metabolite to its original parent form. This work describes a 4 minute LC/MS/MS method that eliminates the need for sample hydrolysis.
Enhanced Resolution of Vitamin D Metabolites Utilizing Chromatographic Selectivity and Novel Sample Preparation Techniques
Analysis of Vitamin D metabolites has continued to be a topic of interest in recent publications, primarily as biomarkers for possible disease states and vitamin sufficiency. While Vitamin D is present in two forms, current ELISA methods cannot distinguish D2 and D3 forms of the vitamin metabolites resulting in a reporting of total 25-hydroxyvitamin D. In this study, an LC/MS method for the analysis of Vitamin D metabolites is expanded to include dihydroxy metabolites along with the epi-homologs. Chromatographic resolution is utilized for the quantitation of hydroxy and dihydroxy Vitamin D2 and D3 metabolites including the isobaric epimers. In addition, sample preparation techniques are evaluated for the impact of biological matrix ionization effects.
Development of an LC-MS/MS Method to Determine Insulin and Synthetic Analogs in Human Plasma
Several synthetic insulin analogs are available for the treatment of diabetes. These analogs differ in some amino acids from endogenous insulin. Routine diagnostic assays for insulin, usually immunoassays on automated platforms, show highly variable results for recombinant insulin analogs. Therefore an LC-MS/MS method for the quantitative determination of endogenous and synthetic insulin forms was developed and validated. This method can be of clinical importance i.e. for patients with unexplained hypoglycemia and can provide extra information for research questions where both endogenous and exogenous insulin(s) are present.
Analysis of Tramadol and Its Three Desmethyl Metabolites in LCMS Using High Resolution and Accurate Mass Mass Spectrometry. Monitoring in Clinical Toxicology
A new LC-MS method for quantitation of tramadol and the three metabolites O-desmethyltramadol, N-desmethyltramadol and O,N-didesmethyltramadol in human plasma is described. Sample preparation was based on liquid-liquid extraction. Mass spectrometric analysis was performed on an Exactive Plus (Thermo ScientificTM) using electrospray ionization. Data were acquired in full scan MS in positive mode at a resolution of 70.000 at m/z 200. The method was successfully validated in the concentration range 10-2000 ng/mL; accuracy, precision and total extraction recovery were also evaluated. This method was applied to quantify the analgesic in plasma and also evaluate metabolites ratios in some clinical intoxication cases.
Serum Aldosterone Measurement by Liquid Chromatography-tandem Mass Spectrometry
We have developed an UPLC–MS/MS method for the quantitation of aldosterone using LLE and SPE prior to RP chromatography. Transitions for aldosterone m/z 359.0 > 189.1 and d7-aldosterone 366.0 > 338.3 were used for monitoring using –ESI mode. Accuracy was verified by analysis of GC-MS certified aldosterone materials (n=3; RFB, Bonn, Germany)(UPLC-MS/MS= 0.96 RFB + 34.1; r2=0.999). A preliminary method comparison with a second LC-MS/MS method (Vancouver, Canada) showed a good correlation (UPLC-MS/MS= 0.98 LC-MS/MS + 15.4 (r2=0.990; n=5). LLOQ was 30 pmol/L and intra- and inter-assay imprecision were <10%. The assay was linear up to 3140 pmol/L.
Overestimation of 25-hydroxyvitamin D3 by Increased Ionisation Efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS Methods Not Separating Both Metabolites
Using an LC-MS/MS method for separate quantification of (3-epi-)25-(OH)D3 and 25(OH)D2 we could show that in LC-MS/MS methods, not resolving 25(OH)D3 from 3-epi-25(OH)D3, the 3-epi-25(OH)D3 contribution not only is overestimated from its co-elution with 25(OH)D3, but that an additional 37% overestimation is caused by differences in ionisation efficiency between both isomers. Due to its increased ionization efficiency, the contribution of the 3-epi-25(OH)D3 metabolite to the total 25(OH)D3 concentration is significantly overestimated in MS methods that do not resolve 3-epi-25(OH)D3 from 25(OH)D3 which may compromise its use in infant samples, known to have significant amounts of 3-epi-25(OH)D3.
Development of an LC-MS/MS Method for Separate Quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in Human Serum
An LC-MS/MS method was developed for simultaneous quantification of (3-epi-)25-(OH)D3 and 25(OH)D2 in human serum using PFP column chromatography followed by SRM registration using +ESI-MS/MS. Accurate measurement was achieved by use of separate calibration curves and SIL-IS for each compound and was checked from measurement of samples with NIST RMP assigned values. Sample preparation consisted of PP followed by SPE. Inter-assay precision was less than 7.5% for all metabolites; LLOQ were 1, 1 and 2 nmol/L and linearity were 1-500, 1-200 and 2-500 nmol/L for 25(OH)D3, 3-epi-25(OH)D3 and 25(OH)D2, respectively. The PFP method showed minimal bias to the NIST RMP.
Development of a Turboflow™-LC-MS/MS Method for Determination of 17-Hydroxyprogesterone in Human Serum
Despite characterized by high rate of positive results the more widespread methods for 17-OHP quantification are the immunometric ones. We have developed a TurboFlow™-LC-MS/MS method using a Vantage triple quadrupole spectrometer (Thermo Scientific) equipped with an atmospheric pressure chemical ionization source. Method was fully validated and results compared with radioimmunoassay (RIA) currently used in our laboratory.
The method was linear from 0.02 ng/mL to 50 ng/mL. Total imprecision was lower than 5%. The Bland-Altman plot indicates an overestimation of RIA method with respect to TurboFlow™-LC-MS/MS method.
The method, is rapid, sensitive and then suitable for routine purpose.
Ultra High Throughput Analysis of Immunosuppressants in Whole Blood by Microflow LC-MS/MS
Rapid turnaround of immunosuppressant samples, as with all therapeutic drug monitoring, is necessary in a clinical research laboratory. Additionally, as financial and environmental concerns become more prevalent, heavy use of solvents for high flow LC-MS/MS methods, particularly those involving online extraction, can be an issue. Microflow LC offers a solution to these concerns, whilst greatly enhancing on column sensitivity and chromatography performance, reducing sample consumption and improving system robustness and uptime. As the sample injection volume is minimized, robustness and reproducibility of the entire method is dramatically increased as matrix effects are significantly reduced.
Simultaneous Determination of 5 Steroids in Serum Using LC-MS/MS for the Diagnosis of Congenital Adrenal Hyperplasia
We developed and validated more specific and selective method for measurement of serum cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstenedione and 17-hydroxyprogesterone using LC-MS/MS. The steroids were extracted with methyl-tert-butyl ether and separated by using Kinetex XB C18 column and Water-methanol gradient as mobile phase. The five steroids were measured in MRM mode with positive electrospray mode. The method showed good precision, specificity, sensitivity and linearity. It is expected that more specific method presented might be a useful tool for the diagnosis and follow up of CAH patients.
Analysis of Aldosterone in Plasma for Clinical Research Using Automated Extraction
Here we evaluate a UPLC/MS/MS method for the measurement of plasma aldosterone for clinical research purposes. An analytically sensitive method was developed using a mixed-mode Solid Phase Extraction (SPE) sorbent in 96-well plate format. Automated extraction was employed, enabling high throughput of samples. Analysis was performed using an ACQUITY UPLC® I-Class system, samples were injected onto a 2.1 x 50 mm Waters ACQUITY UPLC BEH Phenyl column using a water/methanol gradient and quantified with a Waters Xevo® TQ-S mass spectrometer to obtain the optimal sensitivity.
For Research Use Only, Not for Use in Diagnostic Procedures.
25-Hydroxy-Vitamin D- Which Ionization Mode and Column Type Gives the Best Analytical Results
25-Hydroxyvitamin D can be analyzed by liquid chromatography triple quadrupole mass spectrometry with Electrospray or Atmospheric Pressure Chemical Ionization using various column types. LC/MS/MS analytical methods were developed for the quantitation of 25-Hydroxyvitamin D2/D3 by QQQ in ESI and APCI modes to evaluate which was more sensitive and specific. Using protein crash sample preparation, various column types were evaluated and compared based on their chromatographic resolution, separation, matrix effect, robustness and response. The methods achieved the required sensitivity, specificity and capability of quantitating the analytes over a relevant dynamic range. Excellent reproducibility was observed for all compounds (CV < 5%) with calibration curves displaying linearity with an R2 > 0.995. ESI mode with a Poroshell Bonus RP achieved the best overall analytical results.
UPLC-MS/MS Measurement of Triazole Antifungals and Metabolites in Serum
There is growing clinical demand for therapeutic drug monitoring of triazole antifungals and their metabolites, particularly in immunosuppressed populations. Here, we present a simple and rapid stable isotope dilution liquid chromatography tandem mass spectrometry method to measure fluconazole, voriconazole, voriconazole-N-oxide, posaconazole, itraconazole, and hydroxyitraconazole in serum. Simple protein precipitation was followed by reversed phase UPLC coupled to ESI-MS/MS providing a short run time (3 min), with each analyte showing linearity (r2 > 0.99) from 0.01 µg/mL to 10 µg/mL (0.1 to 100 µg/mL for fluconazole), precision (CV < 15% at 7 concentrations) and a high degree of accuracy when cross-compared to reference clinical samples (r2 > 0.98).
Quantitative Androgen Profile in Plasma Using Online SPE in Combination with UPLC Tandem Mass Spectrometry
The use of mass spectrometric based assays for the determination of steroids is finding its way into the clinical laboratory as the bar for steroid testing is continually being raised. Improved accuracyand sensitivity enhance the clinical use of these biomarkers and routinely applicable methods using generic chromatographic procedures are needed. In this light we developed an LC-MS/MS method for the combined quantification of the androgens: progesterone (P), dehydroepiandrosterone (DHEA), 17-á-hydroyprogesterone (17OHP), testosterone (T), androstenedione (A) and dihydrotestosterone (DHT) in plasma with limited hands-on sample preparation.
Total run-time was 7 min. Intra- and inter-assay analytical variation were <10% for all steroids. Linearity in the calibration range for each respective steroid was excellent (r2 >0.99).
25-Hydroxy-Vitamin D- Which Sample Preparation Technique Offers the Best and Most Effective Results
25-Hydroxyvitamin D can be analyzed by liquid chromatography triple quadrupole mass spectrometry. Using a LC/MS/MS analytical method for the quantitation of 25-Hydroxyvitamin D2 and D3 by QQQ, sample preparation techniques such as protein crash, liquid-liquid extraction, supported liquid extraction and solid phase extraction were evaluated and compared based on their ease of use, analyte recovery, post-extraction cleanliness and overall effectiveness. The sample preparation methods achieved the required sensitivity, specificity and capability of quantitating the analytes over a relevant dynamic range. Excellent reproducibility was observed for all compounds (CV < 5%) with calibration curves displaying linearity with an R2 > 0.995. LLE offered the best overall effectiveness as well as cost but the other techniques presented equivalent results and effectiveness.
Determination of 25-hydroxyvitamin D3 and D2 in Plasma Using Online SPE in Combination with UPLC-MS/MS
25-hydroxyvitamin D3 in plasma is the established marker to detect vitamin D deficiency. Several methods have been published to analyze 25-hydroxyvitamin D3 by LC-MS/MS. Most of these methods use protein precipitation or/and offline SPE methods. Here we present a fully automatable method by combing online SPE with UPLC-MS/MS.
Total run-time was 6.5 min, resulting in baseline separation between 25-hydroxyvitamin D3 and D2. Intra- and inter-assay analytical variation were <5% for both vitamins. Linearity in the calibration range for D3 and D2 was excellent (r2 >0.99).
In summary, we have developed a selective and reproducible mass spectrometric analysis for the measurement of 25-hydroyvitamin D3 and D2 in plasma which can be completely automated.
Development of a UPLC-MS/MS Method to Measure Next Generation Antiepileptic Medications in Serum
Next generation antiepileptic medications have revolutionized the treatment of epilepsy as well as other neurological disorders. As with traditional anticonvulsant therapy, however, inter-individual variability in pharmacokinetics has increased the need for therapeutic drug monitoring for effective dosing and decreased toxicity. In this study, we developed a fast and simple stable isotope dilution UPLC-MS/MS method to measure levetiracetam, lamotrigine, gabapentin, zonisamide and topiramate in serum. A total method run time of three minutes allowed adequate separation of all analytes with sensitivity and linearity covering the relevant serum concentration range found in humans.
Evaluation of New PreludeTM SPLC System Coupled with EnduraTM Triple
Quadrupole MS Using Analysis of Testosterone and Cortisol in Biological Matrixes
Growing demand for cost-efficient and easy to use LC-MS solutions has resulted in development of new instrumentation now available to laboratories.
We evaluated the Prelude SPLC system coupled to an Endura triple quadrupole mass spectrometer for the analysis of testosterone and cortisol in plasma and urine, respectively. The Prelude system offers cost efficient methods by providing on-line sample clean up, low solvent consumption, and increased sample throughput with dual
channel multiplexing. Additionally, syringe pumps give reproducible gradients which improve data quality.
Data obtained met analytical laboratory requirements for LOQ, linear range, precision and accuracy. No matrix effects were observed.
Multiplexing Multiple Methods to Maximize Workflow Efficiency in LC-MS
Laboratories
Many research laboratories run several different LC-MS methods in series on a single channel LC-MS system. If the methods involve different columns and mobile phases, the changeover is time consuming, labor intensive and increases the risk of mistakes and
contamination. A four-channel UHPLC system multiplexed into one mass spectrometer permits parallel batches of up to four different methods utilizing unique columns and mobile phases to be completed in a fraction of the time and effort.
Performance Evaluation of Serum 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 Using PerkinElmer MSMS Vitamin D Kit
We evaluated the performance of the recently introduced PerkinElmer vitamin D kit. Accuracy, precision, linearity, LLOQ, recovery and carry-over of MSMS Vitamin D kit were evaluated. The MSMS Vitamin D kit was found to produce intra-assay and inter-assay CV of less than 6% for precision and showed a bias of less than 5%. The vitamin D kit displayed linearity in the range of 25OHD levels of 4.5-150 ng/mL, and the LLOQ for 25OHD was 0.38 ng/mL. The RIA measurements of 25OHD showed a correlation of y=0.9931x+0.2216 (r2=0.74) with LC-MS/MS values. This LC-MS/MS assay of 25OHD3 and 25OHD2 showed an excellent performance from the application of the MSMS Vitamin D kit. Assays using the MSMS Vitamin D kit are considered as more standardized methods that enable quicker and more accurate analysis than other existing methods and help reduce inter-laboratory variation.
Measurement of C3-epimer Form of 25-hydroxyvitamin D3 in Korean Children
Using PerkinElmer MSMS Vitamin D Kit using LC-MS/MS, we established and validated measurement method of C3-epimer-25OHD3(3-EpiD3). We measured 3-EpiD3 in 91 Korean children by this method. This assay showed high accuracy, precision and sensitivity. Measurements of 3-EpiD3 ranged from less than LoQ level(1.30 ng/mL) to 3.35 ng/mL. Proportion of 3-EpiD3 among 25OHD3 ranged from 3% to 38%(median 6%). Before 12 mo of age, high 3-EpiD3 level more than 2.0 ng/mL was often(27%) observed(median 1.6 ng/mL). While after 12mo, 3-EpiD3 decreased overall showing median level of 1.3 ng/mL and high 3-EpiD3 level more than 2.0 ng/mL was rare(5%). We can successfully measure 3-EpiD3 with PerkinElmer MSMS Vitamin D Kit using LC-MS/MS. And 3-EpiD3 level of Korean children was relatively high in infants and it showed decreasing change with time.
Development and Validation of a Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) Assay for Voriconazole
Background: Our institution currently sends serum samples for voriconazole testing to a reference laboratory. Objective: Develop a LC-MS/MS assay for voriconazole. Results: Six calibrators were used (0-10 µg/mL) and the reportable range was 0.1-40 µg/mL. The LOQ=0.1 µg/mL. Extraction recovery was ~69% and ion suppression was ~13%. Run time was 4 minutes. Precision studies yielded CVs <5% at LOQ and <4% through the linear range. Conclusions: An accurate, sensitive and rapid LC-MS/MS assay for voriconazole was developed. Method comparison, cost-analysis and electronic data upload to the laboratory information system studies are ongoing.
Urine Drug Screening by GC-MS/MS and LC-MS/MS
Clinical toxicology services are routinely offered by laboratories and can play an important role in patient management. Our laboratory offers a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to screen urine samples for a diverse group of compounds. Immunoassays are not available for many of the analytes, making it challenging to verify results. Here we report the development and validation of a gas chromatography tandem mass spectrometry method for comprehensive urine drug screening of underivitized samples, which we use to corroborate our LC-MS/MS results.
Method for Drug Screening in Exhaled Breath Using Liquid Chromatography – Tandem Mass Spectrometry
A mass spectrometric multicomponent method for analysis of drugs of abuse in aerosol particles collected from exhaled breath employing a new sampling device was developed and applied in routine. Analytes comprised morphine, 6-acetylmorphine, amphetamine, methamphetamine, benzoylecgonine, cocaine, diazepam, oxazepam and tetrahydrocannabinol. The methods involved a simple sample preparation procedure for eluting drugs with methanol from the collection filter, quantification using deuterated analogues (internal standards), reversed phase chromatography with gradient elution, positive electrospray ionization and monitoring of two product ions per analyte in selected reaction monitoring mode (Thermo Fisher Scientific TSQ Vantage instrument). The measuring range was 6.0–1000.0 pg/sample and the reporting level in routine analysis was set to18 pg/sample for all analytes.
A Method for the Determination of Desomorphine, Heroin, Methadone, Buprenorphine and Metabolites in Urine Using LC/MS QQQ
Analytical measurement of opioid panels commonly include a number of compounds such as heroin, methadone and buprenorphine. Research methods with sufficient flexibility are of value to respond to the need to measure new compounds resulting from clandestine production.
Desomorphine, also known as Krokodil, is an emerging synthetic opioid, sometimes used as a heroin substitute. This poster describes the development of an LC/MS QQQ research method that rapidly quantifies desomorphine together with heroin, buprenorphine, methadone and their metabolites 6-acetylmorphine, norbuprenorphine and EDDP over a concentration range of 1-5000 ng/ml. Precision data, LLOQ and calibration linearity will be reported for each analyte.
Analysis of Cannabinoids in Whole Blood: Above and Beyond State of Art
Since the Decree-law of 22 February 1990, the Cannabis, in any form is classified as a narcotic in France. After consumption, 9-tetrahydrocannabinol (THC), the major active form, is metabolized primarily as 11OH-Δ9-tetrahydrocannabinol (THC-OH) and acid 11nor-Δ9-tetrahydrocannabinol (THC-COOH). THC is detectable within seconds after first inhalation and maximum concentration in blood is obtained in less than 10 minutes. The maximum concentration of THC-OH is generally reached in less than 30 minutes, and THC-COOH in less than 2 hours. The recommended limits of quantification are 0.5 ng/mL for THC and THC-11OH and 2 ng/mL for THC-COOH.
In this workflow, we describe a new approach allowing the quantification of the three analytes respecting the new guidelines of French Analytical Toxicology Society (SFTA) on Cannabis quantitation in whole blood.
Comprehensive Drugs of Abuse Identification in Urine by LC-MSn Combined with MS Spectral Library Matching
Due to the ease of noninvasive sampling, urine has become a sample of choice for drugs-of-abuse identification. Detection is traditionally achieved by immunoassays. Their simplicity and speed enable high-throughput analysis. However, immunoassays cover only a limited number of drugs. Many assays identify only drug classes producing a significant rate of false-positives due to antibody cross-reactivities. Thus, often a secondary, more specific analysis such as LC-MS/MS is required.
Here we present an ion trap LC-MSn-method using full scan MS, MS2 and MS3 spectra from +ESI and -ESI polarities with retention time matching for a broad automated screening based on spectral libraries.
Evaluating ToxFinderTM New Data Processing Software in Targeted Screening Applications Implemented on LC/MS Mass Spectrometers
Targeted screening applications are commonly used in forensic toxicology laboratories. Screening applications utilize all kind of mass spectrometers, each with advantages and limitations. User friendly software that fully utilizes screening data and simplifies data interpretation is a critical component. Here we evaluate new data processing software which supports two screening methods or orbitrap mass spectrometers and one with SRM transitions on triple quadrupole mass spectrometers. The software provides intuitive data processing workflow, rapid data review and custom reporting. It supports requirements for relative retention time and semi-quantitative assessment, as well as output for easy data transfer to laboratory LIMS system.
Rapid and Simple Confirmation and Quantification of 11-Nor-Δ9-Tetrahydrocannabinol-9-Carboxylic Acid (THC-COOH) in Human Urine by 2D-LC-MS/MS
Cannabinoids commonly detected compounds in toxicology screening programs and workplace drug testing. Urine is tested for 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) including its ester-glucuronides. Traditionally this has involved hydrolysis, SPE, derivatisation and GC-MS detection. We sought to replace this with a rapid 2D-LC-MS/MS method. Samples were hydrolysed by NaOH directly in the injection vial, neutralised and injected into the 2D-LC-MS/MS with a total runtime of 2.2 min. The assay was validated over 5-1000 ng/ml with minimal matrix effects. It performed well on external quality control samples and is to be used for >60 samples/day for medical screening and workplace confirmations meeting AS/NZS 4308:2008 standards.
Screening for Psychotropic Medical Drugs in Serum using Ion Trap MS – Customizing a Screening Approach to Specific Needs in the Lab
Prescription volumes of psychotropic medical drugs increased over the last years and so did their occurrence in cases of intoxication or driving under the influence of drugs. The aim of this project was to develop a screening method for these compounds in serum by customizing the ToxtyperTM screening approach. Serum samples fortified with low therapeutic concentrations of 105 compounds and several samples of authentic cases were analyzed using data dependent acquisition of spectra to evaluate the method. For the majority of compounds automatic identification of low therapeutic levels could be shown, making the screening suitable for clinical and forensic applications.
An Automated LC-Ion Trap MS Screening for Synthetic Cannabinoids
Due to the high potency of synthetic cannabinoids, hyphenated and sensitive mass spectrometry is required for the detection of these drugs in biological specimens. This project aims at developing and implementing an automated screening procedure for the detection of synthetic cannabinoids in serum using a liquid chromatography-ion trap-MS system and a spectra library-based approach. In the process of method development a new ion-source type and its effect on the ionization efficiency of the investigated synthetic cannabinoids was evaluated and compared to a conventional ESI-source. All compounds could automatically be identified in human serum at concentrations of 0.5 ng/mL or less.
Comparison of 25-hydroxyvitamin D Assays in Korean with Detectable Ergocalciferol
Among two forms of 25-hydroxyvitamin D, ergocalciferol (D2) is usually not detected in the majority of Korean. This study was to evaluate the performance of two vitamin D immunoassays with samples of detectable D2. Fifty serum samples were analyzed with LC-MS/MS, ADVIA Centaur and Elecsys Vitamin D Total Assay. The mean difference of Centaur and Elecsys were -7.2 ng/mL and +1.1 ng/mL, respectively. The correlation of Elecsys with LC-MS/MS was better (R=0.8635) than that of Centaur (R=0.6234). The correlation of Centaur was inferior compared to previous studies. There might be underestimation of D2 levels in some vitamin D immunoassays.
Development and Validation of a Candidate Reference Method for the Measurement of Serum Cortisol Using Supported Liquid Extraction and UPLC-MS/MS
We present a novel candidate reference method for the measurement of serum cortisol using SLE and UPLC-MS/MS. National quality assurance (QA) schemes have demonstrated that cortisol measurements are inaccurate and display high variability. This method utilises certified reference material to ensure results are traceable to the International System of Units (SI). The analytical quality specifications have been validated against a panel of 34 certified European Reference Materials (ERM-DA451). The excellent accuracy and precision of this method qualify it as a candidate reference method that can be utilised to underpin QA schemes by assigning traceable target values.
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